Methods of treating and predicting non-response to anti-TNF treatment in subjects with inflammatory bowel disease

ABSTRACT

The aspects disclosed herein describe methods of identifying a subject that is non-responsive to anti-TNF therapy. The aspects disclosed herein further provide for a method of selecting a therapy for a subject with Inflammatory Bowel Disease (IBD), and treating the subject with the therapy.

PRIORITY

This application claims priority to U.S. Provisional Application Ser.No. 62/487,971 filed Apr. 20, 2017, the entirety of which is herebyincorporated by reference herein.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. DK046763awarded by National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy created Apr. 18, 2018, isnamed 52388-729_201_SL and is 164,651 bytes in size.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Inflammatory bowel disease (IBD) has two common forms, Crohn's disease(CD) and ulcerative colitis (UC), which are chronic, relapsinginflammatory disorders of the gastrointestinal tract. In 2015, anestimated 1.3% of adults in the United States (3 million) reported beingdiagnosed with either CD or UC.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 depicts in accordance with various embodiments of an aspectprovided herein, genome-wide association with ANCA level in discoverycohort.

FIG. 2 depicts in accordance with various embodiments of an aspectprovided herein, the genomic inflation factor for the genome-wideassociation in the discovery cohort.

FIGS. 3A and 3B depict in accordance with various embodiments of anaspect provided herein, the association of rs5745994 with serum TNFR1and TNFR2 levels. FIG. 3A shows the association of rs5745994 with TNFR1levels. FIG. 3B shows association of rs5745994 with TNFR2 levels.

SUMMARY

It is hypothesized that IBD is caused by an inappropriate immuneresponse to normal enteric flora in genetically susceptible individuals.In addition, there is a higher prevalence of serological responses tomicrobial or autoantigens in subjects diagnosed with IBD. A number ofserological markers have been used in clinical practice, includingatypical perinuclear antineutrophil cytoplasmic antibody (ANCA). Studieshave investigated the potential value of serological markers, like ANCA,in the diagnosis and characterization of IBD.

Anti-tumor necrosis factor (TNF), or TNF-alpha (TNF-α), is a gene thatencodes a multifunctional proinflammatory cytokine implicated in avariety of inflammatory diseases and disorders, including IBD. Severalanti-TNF-alpha blocking strategies have been evaluated in patients withIBD, including infliximab, adalimumab, and certolizumab. Various paststudies have demonstrated that infliximab has the potential to beeffective for the induction and maintenance response and remission insome CD patients. However, the clinical trial data for allanti-TNF-alpha therapies among adult CD patients report that 40% ofpatients do not respond to the induction phase (primary non-responder)and that approximately 40% of those patients who do enter themaintenance phase of the trial lose response over time. Inter-individualvariability in therapeutic response may be best explained by geneticvariability and non-genetic factors, such as serological markers, asthey relate to IBD pathogenesis and mechanism of action of this class oftherapies. The potential clinical and genetic associations betweengenetic risk variants, ANCA, and IBD may provide important insights totreating or predicting anti-TNF nonresponse in patients with IBD.

There is no single or combination diagnostic test that can predict, witha high enough degree of accuracy, patients who will primarily notrespond to anti-TNF therapy. With new treatments available, using risksingle nucleotide polymorphisms (SNPs) disclosed herein in combinationwith ANCA to identifying subjects non-responsive to anti-TNF therapywould be a great test to eliminate expensive delay of effectivetreatment by a different drug, such an inhibitor of a different target,like tumor necrosis family ligand 1 (TL1A).

Aspects disclosed herein are based, at least in part, on these findingsand addresses the need in the art for methods of identifying andtreating a subject that is non-responsive to anti-TNF therapy and amethod of selecting a therapy for a subject with an IBD using TNFRSF1Band ANCA.

In an aspect, the present application provides methods and systems fortreating an inflammatory disease or condition, or fibrostenotic and/orfibrotic disease in a subject comprising with a therapeuticallyeffective amount of a therapeutic agent, provided a level ofantineutrophil cytoplasmic antibody (ANCA), or a presence of a geneticrisk variant comprising a “C” at nucleobase 256 within rs5745994 of agene locus TNFRSF1B, or a combination thereof, is detected in abiological sample obtained from the subject. In some cases, a firstthreshold level of ANCA that is at, or above, 100 ELISA units (EU) isused as an independent indicator that the subject is non-responsive, orsusceptible to non-response, to anti-TNF therapy. In some cases, asecond threshold level of ANCA that is below the first threshold leveland at, or above 50 EU, is an indicator of the subject being, or beingsusceptible to, non-response to anti-TNF therapy if either (i) adecrease in circulating TNFR2 levels and/or (ii) a presence of a geneticrisk variant or SNP at the TNFRSF1B gene locus is detected in thebiological sample obtained from the subject. In some cases, thetherapeutic agent inhibits the activity or expression of gene expressionproducts from genes comprising TL1A, JAK1, GPR35, ADCY7, IFNG, TNFSF8,PFKFB3, SKAP2, GPR65, SPRED2, IL18R1, and/or GSDMB. In some cases, thetherapeutic agent comprises an anti-TL1A antibody.

Further provided are methods of detecting, and kits for detecting, ANCAlevels, the presence of the genetic risk variant or genotype, and/orTNFR2 levels in a biological sample obtained from a subject with aninflammatory disease or condition, or fibrostenotic and/or fibroticdisease. Such genetic risk variants or genotypes comprise singlenucleotide polymorphisms (SNPs) in rs5745994 and/or rs11757159.Exemplary detection methods involve hybridization assays using nucleicacid probes specific for the SNPs. In some cases, the methods ofdetecting, and kits for detecting, are used to treat the subject asdisclosed herein.

In one aspect, provided herein are methods of treating a subject with aninflammatory disease or condition, or fibrostenotic and/or fibroticdisease, comprising administering to the subject a therapeuticallyeffective amount of a therapeutic agent, provided a level ofantineutrophil cytoplasmic antibody (ANCA), or a presence of a geneticrisk variant comprising a risk allele at nucleobase 256 within rs5745994of a gene locus TNFRSF1B (SEQ ID NO: 1), or a combination thereof, isdetected in a biological sample obtained from the subject. In someembodiments the genetic risk variant comprises a “C” at nucleobase 501within rs11757159 of gene locus HLA-DRB6 (SEQ ID NO: 2). In someembodiments, the genetic risk variant comprises a “C” at nucleobase 256within rs5745994 of a gene locus TNFRSF1B (SEQ ID NO: 1). In someembodiments, a single copy of the genetic risk variant is detected. Insome embodiments a single copy of the genetic risk variant confers aheterozygous risk genotype. In some embodiments, two copies of thegenetic risk variant are detected. In some embodiments, two copies ofthe genetic risk variant confers a homozygous risk genotype. In someembodiments, a decreased level of TNFR2 is detected in the biologicalsample obtained from the subject, compared to a reference value obtainedfrom an individual who is not a carrier of the genetic risk variant. Insome embodiments, the level of ANCA is at or above a first thresholdlevel comprising 100 ELISA units (EU). In some embodiments, a level ofANCA that is at, or above, the first threshold level detected in thebiological sample obtained from the subject is an independent indicatorthat the subject is non-responsive, or is susceptible to non-response,to anti-TNF therapy. In some embodiments, the level of ANCA is lowerthan the first threshold level and higher than a second threshold levelcomprising 50 EU. In some embodiments, the level of ANCA is between 50and 60 EU. In some embodiments, the ANCA level is between 60 and 70 EU.In some embodiments, the ANCA level is between 70 and 80 EU. In someembodiments, the ANCA level is between 80 and 90 EU. In someembodiments, the ANCA level is above 90 EU and below 100 EU. In someembodiments, a level of ANCA below 100 EU and at or above 50 EU, thepresence of the genetic risk variant, and a decreased level of TNFR2,detected in the biological sample obtained from the subject areindicative that the subject is non-responsive, or is susceptible tonon-response, to anti-TNF therapy. In some embodiments, the presence ofthe genetic risk variant detected in the biological sample obtained fromthe subject is indicative that the subject is non-responsive, or issusceptible to non-response, to anti-TNF therapy. In some embodiments, alevel of ANCA below 100 EU and at or above 50 EU and the presence of thegenetic risk variant detected in the biological sample obtained from thesubject are indicative that the subject is non-responsive, or issusceptible to non-response, to anti-TNF therapy. In some embodiments, alevel of ANCA below 100 EU and at or above 50 EU and a decreased levelof TNFR2, detected in the biological sample obtained from the subjectare indicative that the subject is non-responsive, or is susceptible tonon-response, to anti-TNF therapy. In some embodiments, the presence ofthe genetic risk variant and a decreased level of TNFR2 detected in abiological sample obtained from the subject are indicative that thesubject is non-responsive, or is susceptible to non-response, toanti-TNF therapy. In some embodiments, the level of ANCA is detectedusing an assay comprising an anti-ANCA antibody. In some embodiments,the assay is an enzyme-linked immunosorbent assay (ELISA). In someembodiments, the ELISA is an indirect ELISA. In some embodiments, theELISA is a fixed leukocyte ELISA. In some embodiments, the ELISA is afixed neutrophil ELISA. In some embodiments, the inflammatory disease orcondition, or fibrostenotic and/or fibrotic disease, comprisesinflammatory bowel disease (IBD), Crohn's disease (CD), perianal Crohn'sdisease (pCD), ulcerative colitis (UC), rheumatoid arthritis, multiplesclerosis, psoriasis, chronic colitis, pancreatitis, leukopenia, colonicfibrosis, primary sclerosing cholangitis, progressive systemicsclerosis, chronic asthma, or fibrostenosis of a small and/or largeintestine, or a combination thereof. In some embodiments, the geneticrisk variant is detected by contacting the biological sample obtainedfrom the subject with a nucleic acid sequence capable of hybridizing to10 contiguous nucleobases of SEQ ID NO. 1 spanning nucleobase 256 understandard hybridization conditions. In some embodiments, the standardhybridization conditions comprise an annealing temperature between about30° C. and about 65° C. In some embodiments, the therapeutic agentcomprises an agonist or an antagonist of gene expression products fromgenes comprising TL1A, JAK1, GPR35, ADCY7, IFNG, TNFSF8, PFKFB3, SKAP2,GPR65, SPRED2, IL18R1, and/or GSDMB. In some embodiments, thetherapeutic agent comprises an antibody, small molecule, or stem celltherapy. In some embodiments, the therapeutic agent comprises anantibody that inhibits the expression or activity of TL1A, JAK1, GPR35,ADCY7, IFNG, TNFSF8, PFKFB3, SKAP2, GPR65, SPRED2, IL18R1, and/or GSDMB.In some embodiments, the therapeutic agent comprises an anti-TLAantibody. In some embodiments, the therapeutic is second-line to ananti-TNF therapy, a steroid, and/or an immunomodulator.

In another aspect, the present application provides methods comprising:assaying to detect in a biological sample obtained from a subject withan inflammatory disease or condition, or fibrostenotic and/or fibroticdisease a level of antineutrophil cytoplasmic antibody (ANCA), apresence of a genetic risk variant comprising a risk allele atnucleobase 256 within rs5745994 of a gene locus TNFRSF1B, or acombination thereof; identifying the subject as being non-responsive toanti-TNF therapy, or susceptible to non-response to anti-TNF therapy,provided (i) a level of ANCA that is at or above a first threshold levelcomprising 100 ELISA units (EU), or (ii) a level of ANCA that is lowerthan the first threshold level and above a second threshold levelcomprising about 50 EU, and the presence of the genetic risk variant,are detected in the biological sample obtained from the subject. In someembodiments the genetic risk variant comprises a “C” at nucleobase 501within rs11757159 of gene locus HLA-DRB6. In some embodiments, thegenetic risk variant comprises a “C” at nucleobase 256 within rs5745994of a gene locus TNFRSF1B (SEQ ID NO: 1). In some embodiments, a singlecopy of the genetic risk variant is detected. In some embodiments asingle copy of the genetic risk variant confers a heterozygous riskgenotype. In some embodiments, two copies of the genetic risk variantare detected. In some embodiments, two copies of the genetic riskvariant confers a homozygous risk genotype. In some embodiments, themethods further comprise assaying to detect a decreased level of TNFR2is detected in the biological sample obtained from the subject, ascompared to a reference value obtained from an individual who is not acarrier of the genetic risk variant. In some embodiments, the level ofANCA is at or above a first threshold level comprising 100 ELISA units(EU). In some embodiments, a level of ANCA that is at, or above, thefirst threshold level detected in the biological sample obtained fromthe subject is an independent indicator that the subject isnon-responsive, or is susceptible to non-response, to anti-TNF therapy.In some embodiments, the level of ANCA is lower than the first thresholdlevel and higher than a second threshold level comprising 50 EU. In someembodiments, the level of ANCA is between 50 and 60 EU. In someembodiments, the ANCA level is between 60 and 70 EU. In someembodiments, the ANCA level is between 70 and 80 EU. In someembodiments, the ANCA level is between 80 and 90 EU. In someembodiments, the ANCA level is above 90 EU and below 100 EU. In someembodiments, a level of ANCA below 100 EU and at or above 50 EU, thepresence of the genetic risk variant, and a decreased level of TNFR2,detected in the biological sample obtained from the subject areindicative that the subject is non-responsive, or is susceptible tonon-response, to anti-TNF therapy. In some embodiments, the presence ofthe genetic risk variant detected in the biological sample obtained fromthe subject is indicative that the subject is non-responsive, or issusceptible to non-response, to anti-TNF therapy. In some embodiments, alevel of ANCA below 100 EU and at or above 50 EU and the presence of thegenetic risk variant detected in the biological sample obtained from thesubject are indicative that the subject is non-responsive, or issusceptible to non-response, to anti-TNF therapy. In some embodiments, alevel of ANCA below 100 EU and at or above 50 EU and a decreased levelof TNFR2, detected in the biological sample obtained from the subjectare indicative that the subject is non-responsive, or is susceptible tonon-response, to anti-TNF therapy. In some embodiments, the presence ofthe genetic risk variant and a decreased level of TNFR2 detected in abiological sample obtained from the subject are indicative that thesubject is non-responsive, or is susceptible to non-response, toanti-TNF therapy. In some embodiments, methods further comprise treatingthe subject with a therapeutically effective amount of a therapeuticagent, provided the subject is identified as being non-responsive toanti-TNF therapy, or susceptible to non-response to anti-TNF therapy. Insome embodiments, the therapeutic agent comprises an agonist or anantagonist of gene expression products from genes comprising TL1A, JAK1,GPR35, ADCY7, IFNG, TNFSF8, PFKFB3, SKAP2, GPR65, SPRED2, IL18R1, and/orGSDMB. In some embodiments, the therapeutic agent comprises an antibody,small molecule, or stem cell therapy. In some embodiments, thetherapeutic agent comprises an antibody that inhibits the expression oractivity of TL1A, JAK1, GPR35, ADCY7, IFNG, TNFSF8, PFKFB3, SKAP2,GPR65, SPRED2, IL18R1, and/or GSDMB. In some embodiments, thetherapeutic agent comprises an anti-TLA antibody. In some embodiments,the therapeutic is second-line to an anti-TNF therapy, a steroid, and/oran immunomodulator. In some embodiments, the inflammatory disease orcondition, or fibrostenotic and/or fibrotic disease, comprisesinflammatory bowel disease (IBD), Crohn's disease (CD), perianal Crohn'sdisease (pCD), ulcerative colitis (UC), rheumatoid arthritis, multiplesclerosis, psoriasis, chronic colitis, pancreatitis, leukopenia, colonicfibrosis, primary sclerosing cholangitis, progressive systemicsclerosis, chronic asthma, or fibrostenosis of a small and/or largeintestine, or a combination thereof. In some embodiments, the level ofANCA is detected using an assay comprising an anti-ANCA antibody. Insome embodiments, the assay is an enzyme-linked immunosorbent assay(ELISA). In some embodiments, the ELISA is an indirect ELISA. In someembodiments, the ELISA is a fixed leukocyte ELISA. In some embodiments,the ELISA is a fixed neutrophil ELISA. In some embodiments, the geneticrisk variant is detected by contacting the biological sample obtainedfrom the subject with a nucleic acid sequence capable of hybridizing to10 contiguous nucleobases of SEQ ID NO. 1 spanning nucleobase 256 understandard hybridization conditions. In some embodiments, the standardhybridization conditions comprise an annealing temperature between about30° C. and about 65° C.

In another aspect, the present application provides kits comprising acomposition comprising at least 10 but less than 50 contiguousnucleobase residues of SEQ ID NO. 1, wherein the contiguous nucleobaseresidues comprise the nucleobase at position 256 of SEQ ID NO. 1, andwherein the contiguous nucleobase residues are connected to a detectablemolecule comprising a fluorophore, and a primer pair configured tohybridize to 10 contiguous nucleobases of SEQ ID NO. 1 spanningnucleobase 256. In some embodiments, the kit further comprises a fixedenzyme-linked immunosorbent assay (fixed-ELISA) comprising an anti-ANCAantibody. In some embodiments, the fixed-ELISA is a fixed leukocyteELISA. The some embodiments, the ELISA is a fixed neutrophil ELISA. Insome embodiments the kit is used for the treatment of a subject who hasan inflammatory disease or condition, or fibrostenotic and/or fibroticdisease with the therapeutic agent disclosed herein. In someembodiments, treatment of the subject comprises administering to thesubject a therapeutically effect amount of a therapeutic agent. In someembodiments, the therapeutic agent comprises an agonist or an antagonistof gene expression products from genes comprising TL1A, JAK1, GPR35,ADCY7, IFNG, TNFSF8, PFKFB3, SKAP2, GPR65, SPRED2, IL18R1, and/or GSDMB.In some embodiments, the therapeutic agent comprises an antibody, smallmolecule, or stem cell therapy. In some embodiments, the therapeuticagent comprises an antibody that inhibits the expression or activity ofTL1A, JAK1, GPR35, ADCY7, IFNG, TNFSF8, PFKFB3, SKAP2, GPR65, SPRED2,IL18R1, and/or GSDMB. In some embodiments, the therapeutic agentcomprises an anti-TLA antibody. In some embodiments, the therapeutic issecond-line to an anti-TNF therapy, a steroid, and/or animmunomodulator.

Certain Terminologies

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of aspects provided herein. Other features and advantages ofaspects provided herein will become apparent from the following detaileddescription, taken in conjunction with the accompanying drawings, whichillustrate, by way of example, various features of embodiments ofaspects provided herein. Indeed, aspects provided herein are in no waylimited to the methods and materials described. For convenience, certainterms employed herein, in the specification, examples and appendedclaims are collected here.

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments, and are not intended to limit theaspects provided herein, because the scope of the aspects providedherein is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which aspectsprovided herein belong.

Non-limiting examples of “Biological sample” as used herein means anybiological material from which nucleic acids and/or proteins can beobtained. As non-limiting examples, the term encompasses whole blood,peripheral blood, plasma, serum, saliva, mucus, urine, semen, lymph,fecal extract, cheek swab, cells or other bodily fluid or tissue,including but not limited to tissue obtained through surgical biopsy orsurgical resection. In various embodiments, the sample comprises tissuefrom the large and/or small intestine. In various other embodiments, thelarge intestine sample comprises the cecum, colon (the ascending colon,the transverse colon, the descending colon, and the sigmoid colon),rectum and/or the anal canal. In yet other embodiments, the smallintestine sample comprises the duodenum, jejunum, and/or the ileum.Alternatively, a sample can be obtained through primary patient derivedcell lines, or archived patient samples in the form of preservedsamples, or fresh frozen samples.

“Treatment” and “treating” as used herein refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition,prevent the pathologic condition, pursue or obtain good overallsurvival, or lower the chances of the individual developing thecondition even if the treatment is ultimately unsuccessful. Those inneed of treatment include those already with the condition as well asthose prone to have the condition or those in whom the condition is tobe prevented.

“SNP” as used herein means single nucleotide polymorphism.

“Genetic risk variant” as used herein refers to an allele, whosepresence in a polynucleotide sequence is associated with a disease orcondition, or susceptibility to developing the disease or condition.Non-limiting examples of diseases or conditions include, inflammatorydisease or condition, or fibrostenotic and/or fibrotic disease,comprises inflammatory bowel disease (IBD), Crohn's disease (CD),perianal Crohn's disease (pCD), ulcerative colitis (UC), rheumatoidarthritis, multiple sclerosis, psoriasis, chronic colitis, pancreatitis,leukopenia, colonic fibrosis, primary sclerosing cholangitis,progressive systemic sclerosis, chronic asthma, fibrostenosis of a smalland/or large intestine, anti-TNF non-response, leukopenia, andpancreatitis.

“IBD”, “CD” and “UC” as used herein refer to Inflammatory Bowel Disease,Crohn's Disease, and Ulcerative Colitis, respectively.

As used herein, “ANCA” means anti-neutrophil cytoplasmic antibodies.

The terms “increased,” or “increase” are used herein to generally meanan increase by a statically significant amount; in some embodiments, theterms “increased,” or “increase,” mean an increase of at least 10% ascompared to a reference level, for example an increase of at least about10%, at least about 20%, or at least about 30%, or at least about 40%,or at least about 50%, or at least about 60%, or at least about 70%, orat least about 80%, or at least about 90% or up to and including a 100%increase or any increase between 10-100% as compared to a referencelevel. Other examples of “increase” include an increase of at least2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least50-fold, at least 100-fold, at least 1000-fold or more as compared to areference level.

The terms, “decreased” or “decrease” are used herein generally to mean adecrease by a statistically significant amount. In some embodiments,“decreased” or “decrease” means a reduction by at least 10% as comparedto a reference level, for example a decrease by at least about 20%, orat least about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% decrease (e.g., absentlevel or non-detectable level as compared to a reference level), or anydecrease between 10-100% as compared to a reference level. In thecontext of a marker or symptom, by these terms is meant a statisticallysignificant decrease in such level. The decrease can be, for example, atleast 10%, at least 20%, at least 30%, at least 40% or more, and ispreferably down to a level accepted as within the range of normal for anindividual without a given disease.

“Non-response” or “non-responsive” as used herein, refers to a conditioncharacterized by a subject not responding to the induction of a therapy(primary non-response) or a condition characterized by the subjectlosing response to the therapy during the treatment (secondary loss ofresponse). In some embodiments, the therapy may include any therapeuticagent or drug therapy used in the “treating” or “treatment” of asubject, as disclosed herein.

“Susceptible” or “susceptibility” to a disease or condition as usedherein, refers to a condition of the subject characterized by anincreased likelihood to develop the disease or condition, as compared toan individual who is not determined to be susceptible to the disease orcondition. In some embodiments, the condition comprises non-response toanti-TNF therapy. In some embodiments, susceptibility to non-response toanti-TNF therapy in a subject means the subject will more likely thannot develop non-response to anti-TNF therapy, if an anti-TNF therapy isadministered to the subject.

DETAILED DESCRIPTION

In one aspect, provided herein, are methods of obtaining a biologicalsample from a subject with an inflammatory disease or condition orfibrostenotic and/or fibrotic disease, and assaying the sample to detectlevel of antineutrophil cytoplasmic antibody (ANCA), or a presence of arisk allele, “C” at nucleobase 256 within rs5745994 of a gene locusTNFRSF1B, or a decreased level of circulating TNFR2, or a combinationthereof, which is indicative of non-response to anti-TNF therapy in thesubject. In one aspect, provided herein, are methods of treating theinflammatory disease or condition, or fibrostenotic and/or fibroticdisease, in the subject with therapeutic agent, provided the subject isidentified as being non-responsive to anti-TNF therapy. In one aspect,provided herein, are compositions and kits used to identify non-responseto anti-TNF therapy in a subject. In some embodiments, the therapeuticagent is a second-line therapy, comprising an anti-TL1A therapy. In someembodiments, the inflammatory disease comprises Inflammatory BowelDisease (IBD).

Methods of Treating an Inflammatory Disease or Condition orFibrostenotic and/or Fibrotic Disease

In one aspect, provided herein are methods of treating an inflammatorydisease or condition or fibrostenotic and/or fibrotic disease, in asubject. In some embodiments, the subject is a mammal. In someembodiments, the subject is a human. In some embodiments, theinflammatory condition or disease comprises a condition that involveschronic inflammation of the body caused by pathogens, viruses, foreignbodies or overactive immune responses. Non-limiting examples ofinflammatory conditions include, but are not limited to, inflammatorybowel disease (IBD), Crohn's disease (CD), perianal Crohn's disease(pCD), ulcerative colitis (UC), rheumatoid arthritis, multiplesclerosis, psoriasis, chronic colitis, pancreatitis, leukopenia, or acombination thereof. In some embodiments, the fibrostenotic and/orfibrotic disease comprises colonic fibrosis, primary sclerosingcholangitis, progressive systemic sclerosis, chronic asthma, orfibrostenosis of a small and/or large intestine. In some embodiments,the subject is susceptible to, or is inflicted with, thiopurinetoxicity, or a disease caused by thiopurine toxicity (such aspancreatitis or leukopenia). In further embodiments provided, thesubject is non-responsive to a therapy comprising anti-TNF alphatherapy, anti-a4-b7 therapy (vedolizumab), anti-IL12p40 therapy(ustekinumab), Thalidomide, or Cytoxin.

Therapeutic Agent

In one aspect, provided herein are methods of treating an inflammatorydisease or condition or fibrostenotic and/or fibrotic disease in asubject by administering a therapeutically effective amount of atherapeutic agent to the subject. In some embodiments, the therapeuticagent is second-line to a first therapy, or therapies, comprising ananti-TNF therapy, a steroid, and/or an immunomodulator. A second-linetherapy is a therapy that is administered to a subject who (i) does notrespond to the induction of a first therapy (e.g., “primarynon-response”), or is predicted to experience primary non-response, or(ii) experiences loss of response to the first therapy during thetreatment (e.g., “secondary loss of response”). In some embodiments, thetherapeutic agent is an antibody, small molecule, siRNA/short hairpinRNA, peptide, vaccine, or cell-based therapy.

In some embodiments, the therapeutic agent is an immunosuppressant, orfrom a class of drugs that suppress, or reduce, the strength of theimmune system. In some embodiments, the immunosuppressant is anantibody. In some embodiments, the immunosuppressant is a smallmolecule. Non-limiting examples of immunosuppressant therapeutic agentsinclude STELARA® (ustekinumab), azathioprine (AZA), 6-mercaptopurine(6-MP), methotrexate, and cyclosporin A. (CsA). In some embodiments, thetherapeutic agent is a selective anti-inflammatory drug, or from a classof drugs that specifically target pro-inflammatory molecules in thebody. The pro-inflammatory molecules may comprise cytokines, celltrafficking molecules, and/or molecules involved in the innate oradaptive immune responses. The anti-inflammatory drug may comprise anantibody. The anti-inflammatory drug may comprise a small molecule.Non-limiting examples of anti-inflammatory drugs include ENTYVIO®(vedolizumab), corticosteroids, aminosalicylates, mesalamine, COLAZAL®(balsalazide), DIPENTUM® (olsalazine), anti-IL17A (secukinumab),anti-IL13 (tralokinumab and anrukinzumab), MAdCAM-1 (PF-00547659),anti-ICAM (alicaforsen), anti-IL12p40 (ustekinumab), briakinumab(ABT-874), anti-IL23p19 (MED2070), SMAD7-inhibitor (mongersen),modulators of TGFB1, S1PR agonists, anti-TL1A, anti-IL6, anti-IL6R,gp130-Fc, and cirsilineol.

In some embodiments, the therapeutic agent comprises a stem celltherapy. The stem cell therapy may be embryonic or somatic stem cells.The stem cells may be isolated from a donor (allogeneic) or isolatedfrom the subject (autologous). The stem cells may be expandedadipose-derived stem cells (eASCs), hematopoietic stem cells (HSCs),mesenchymal stem (stromal) cells (MSCs), regulatory T cells (Tregs), orinduced pluripotent stem cells (iPSCs) derived from the cells of thesubject. The stem cell therapy may specifically target any of the geneexpression products disclosed herein. The stem cell therapy may be animmunosuppressant, or anti-inflammatory. In some embodiments, the stemcell therapy comprises Cx601/Alofisel® (darvadstrocel).

In some embodiments, the therapeutic agent comprises a small molecule.The small molecule may be used to treat inflammatory diseases orconditions, or fibrostenotic or fibrotic disease. In some embodiments,the small molecule is an agonist. An agonist is a therapeutic agent thatcauses action in the target molecule. In some embodiments, the smallmolecule is an antagonist. An antagonist is a therapeutic agent thatblocks the action of the target molecule. Non-limiting examples of smallmolecules include OTEXLA® (apremilast), alicaforsen, and ozanimod(RPC-1063).

In some embodiments, the therapeutic agent comprises an agonist. In someembodiments, the therapeutic agent comprises an antagonist. In someembodiments, the therapeutic agent comprises a small molecule. In someembodiments, the therapeutic agent comprises an antibody. Thetherapeutic agent may comprise an agonist or an antagonist of TL1A,JAK1, GPR35, ADCY7, IFNG, TNFSF8, PFKFB3, SKAP2 GPR65, SPRED2, IL18R1,GSDMB, or gene expression products from genes implicated in thepathogenesis of inflammatory, fibrotic, or fibrostenotic disease. Theantagonist may inhibit the activity or expression of TL1A, JAK1, GPR35,ADCY7, IFNG, TNFSF8, PFKFB3, SKAP2 GPR65, SPRED2, IL18R1, GSDMB. Thetherapeutic agent may be an allosteric modulator of TL1A, JAK1, GPR35,ADCY7, IFNG, TNFSF8, PFKFB3, SKAP2 GPR65, SPRED2, IL18R1, GSDMB, or geneexpression products from genes implicated in the pathogenesis ofinflammatory, fibrotic, or fibrostenotic disease. Non-limiting examplesof JAK1 inhibitors include ruxolitinib (INCB018424), s-ruxolitinib(INCB018424), baricitinib (LY3009104, INCB028050), filgotinib(GLPG0634), momelotinib (CYT387), cerdulatinib (PRT062070, PRT2070),LY2784544, NVP-BSK805, 2HC1, Tofacitinib (CP-690550,tasocitinib), XL019,pacritinib (SB1518), tofacitinib, or ZM 39923 HCl. In some embodiments,inhibitors of TNFSF8 include anti-CD30L and anti-CD30 therapies. In someembodiments, inhibitors of PFKFB3 include1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK 15).

In some embodiments, the therapeutic agent is an inhibitor of CD30Lexpression or activity. In some embodiments, the inhibitor of CD30Lcomprises an anti-CD30L antibody. The anti-CD30L may comprise a heavychain comprising three complementarity-determining regions: HCDR1,HCDR2, and HCDR3; and a light chain comprising threecomplementarity-determining regions: LCDR1, LCDR2, and LCDR3. In someembodiments, the anti-CD30L antibody comprises a HCDR1 comprising SEQ IDNO: 186, a HCDR2 comprising SEQ ID NO: 187, a HCDR3 comprising SEQ IDNO: 188, a LCDR1 comprising SEQ ID NO: 189, a LCDR2 comprising SEQ IDNO: 190, and a LCDR3 comprising SEQ ID NO: 191.

In some embodiments, the anti-CD30L antibody comprises a HCDR1comprising SEQ ID NO: 192, a HCDR2 comprising SEQ ID NO: 193, a HCDR3comprising SEQ ID NO: 194, a LCDR1 comprising SEQ ID NO: 195, a LCDR2comprising SEQ ID NO: 196, and a LCDR3 comprising SEQ ID NO: 197.

In some embodiments, the anti-CD30L antibody comprises a HCDR1comprising SEQ ID NO: 198, a HCDR2 comprising SEQ ID NO: 199, a HCDR3comprising SEQ ID NO: 200, a LCDR1 comprising SEQ ID NO: 201, a LCDR2comprising SEQ ID NO: 202, and a LCDR3 comprising SEQ ID NO: 203.

In some embodiments, the anti-CD30L antibody comprises a HCDR1comprising SEQ ID NO: 204, a HCDR2 comprising SEQ ID NO: 205, a HCDR3comprising SEQ ID NO: 206, a LCDR1 comprising SEQ ID NO: 207, a LCDR2comprising SEQ ID NO: 208, and a LCDR3 comprising SEQ ID NO: 209.

In some embodiments, the anti-CD30L antibody comprises a HCDR1comprising SEQ ID NO: 210, a HCDR2 comprising SEQ ID NO: 211, a HCDR3comprising SEQ ID NO: 212, a LCDR1 comprising SEQ ID NO: 213, a LCDR2comprising SEQ ID NO: 214, and a LCDR3 comprising SEQ ID NO: 215.

In some embodiments, the anti-CD30L antibody comprises a HCDR1comprising SEQ ID NO: 216, a HCDR2 comprising SEQ ID NO: 217, a HCDR3comprising SEQ ID NO: 218, a LCDR1 comprising SEQ ID NO: 219, a LCDR2comprising SEQ ID NO: 220, and a LCDR3 comprising SEQ ID NO: 221.

In some cases, the anti-CD30L antibody comprises a heavy chain (HC)variable domain comprising SEQ ID NO: 222 and a light chain (LC)variable domain comprising SEQ ID NO: 223. In some cases, the anti-CD30Lantibody comprises a heavy chain (HC) variable domain comprising SEQ IDNO: 224 and a light chain (LC) variable domain comprising SEQ ID NO:225. In some cases, the anti-CD30L antibody comprises a heavy chain (HC)variable domain comprising SEQ ID NO: 226 and a light chain (LC)variable domain comprising SEQ ID NO: 227. In some cases, the anti-CD30Lantibody comprises a heavy chain (HC) variable domain comprising SEQ IDNO: 228 and a light chain (LC) variable domain comprising SEQ ID NO:229. In some cases, the anti-CD30L antibody comprises a heavy chain (HC)variable domain comprising SEQ ID NO: 230 and a light chain (LC)variable domain comprising SEQ ID NO: 231. In some cases, the anti-CD30Lantibody comprises a heavy chain (HC) variable domain comprising SEQ IDNO: 232 and a light chain (LC) variable domain comprising SEQ ID NO:240. In some cases, the anti-CD30L antibody comprises a heavy chain (HC)variable domain comprising SEQ ID NO: 233 and a light chain (LC)variable domain comprising SEQ ID NO: 240. In some cases, the anti-CD30Lantibody comprises a heavy chain (HC) variable domain comprising SEQ IDNO: 234 and a light chain (LC) variable domain comprising SEQ ID NO:240. In some cases, the anti-CD30L antibody comprises a heavy chain (HC)variable domain comprising SEQ ID NO: 235 and a light chain (LC)variable domain comprising SEQ ID NO: 240. In some cases, the anti-CD30Lantibody comprises a heavy chain (HC) variable domain comprising SEQ IDNO: 236 and a light chain (LC) variable domain comprising SEQ ID NO:240. In some cases, the anti-CD30L antibody comprises a heavy chain (HC)variable domain comprising SEQ ID NO: 237 and a light chain (LC)variable domain comprising SEQ ID NO: 240. In some cases, the anti-CD30Lantibody comprises a heavy chain (HC) variable domain comprising SEQ IDNO: 238 and a light chain (LC) variable domain comprising SEQ ID NO:240. In some cases, the anti-CD30L antibody comprises a heavy chain (HC)variable domain comprising SEQ ID NO: 239 and a light chain (LC)variable domain comprising SEQ ID NO: 240.

In some embodiments, the therapeutic agent is an inhibitor of TL1Aexpression or activity. In some embodiments, the inhibitor of TL1Aexpression or activity is effective to inhibit TL1A-DR3 binding. In someembodiments, the inhibitor of TL1A expression or activity comprises anallosteric modulator of TL1A. An allosteric modulator of TL1A mayindirectly influence the effects TL1A on DR3, or TR6/DcR3 on TL1A orDR3. The inhibitor of TL1A expression or activity may be a directinhibitor or indirect inhibitor. Non-limiting examples of an inhibitorof TL1A expression include RNA to protein TL1A translation inhibitors,antisense oligonucleotides targeting the TNFSF15 mRNA (such as miRNAs,or siRNA), epigenetic editing (such as targeting the DNA-binding domainof TNFSF15, or post-translational modifications of histone tails and/orDNA molecules). Non-limiting examples of an inhibitor of TL1A activityinclude antagonists to the TL1A receptors, (DR3 and TR6/DcR3),antagonists to TL1A antigen, and antagonists to gene expression productsinvolved in TL1A mediated disease. Antagonists as disclosed herein, mayinclude, but are not limited to, an anti-TL1A antibody, ananti-TL1A-binding antibody fragment, or a small molecule. The smallmolecule may be a small molecule that binds to TL1A or DR3. Theanti-TL1A antibody may be monoclonal or polyclonal. The anti-TL1Aantibody may be humanized or chimeric. The anti-TL1A antibody may be afusion protein. The anti-TL1A antibody may be a blocking anti-TL1Aantibody. A blocking antibody blocks binding between two proteins, e.g.,a ligand and its receptor. Therefore, a TL1A blocking antibody includesan antibody that prevents binding of TL1A to DR3 and/or TR6/DcR3receptors. In a non-limiting example, the TL1A blocking antibody bindsto DR3. In another example, the TL1A blocking antibody binds to DcR3. Insome cases, the TL1A antibody is an anti-TL1A antibody that specificallybinds to TL1A. The anti-TL1A antibody may comprise one or more of theantibody sequences of Table 1 and/or Table 2. The anti-DR3 antibody maycomprise an amino acid sequence that is at least 85% identical to anyone of SEQ ID NOs:152-164 and an amino acid sequence that is at least85% identical to any one of SEQ ID NOs:165-169. The anti-DR3 antibodymay comprise an amino acid sequence comprising the HCDR1, HCDR2, HCDR3domains of any one of SEQ ID NOs:152-164 and the LCDR1, LCDR2, and LCDR3domains of any one of SEQ ID NOs:165-169.

In some embodiments, an anti-TL1A antibody comprises a heavy chaincomprising three complementarity-determining regions: HCDR1, HCDR2, andHCDR3; and a light chain comprising three complementarity-determiningregions: LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-TL1Aantibody comprises a HCDR1 comprising SEQ ID NO: 3, a HCDR2 comprisingSEQ ID NO: 4, a HCDR3 comprising SEQ ID NO: 5, a LCDR1 comprising SEQ IDNO: 6, a LCDR2 comprising SEQ ID NO: 7, and a LCDR3 comprising SEQ IDNO: 8. In some cases, the anti-TL1A antibody comprises a heavy chain(HC) variable domain comprising SEQ ID NO: 9 and a light chain (LC)variable domain comprising SEQ ID NO: 10.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 11, a HCDR2 comprising SEQ ID NO: 12, a HCDR3 comprising SEQID NO: 13, a LCDR1 comprising SEQ ID NO: 14, a LCDR2 comprising SEQ IDNO: 15, and a LCDR3 comprising SEQ ID NO: 16. In some cases, theanti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 17 and a light chain (LC) variable domaincomprising SEQ ID NO: 18.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 19, a HCDR2 comprising SEQ ID NO: 20, a HCDR3 comprising SEQID NO: 21, a LCDR1 comprising SEQ ID NO: 22, a LCDR2 comprising SEQ IDNO: 23, and a LCDR3 comprising SEQ ID NO: 24. In some cases, theanti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 25 and a light chain (LC) variable domaincomprising SEQ ID NO: 26.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 27, a HCDR2 comprising SEQ ID NO: 28, a HCDR3 comprising SEQID NO: 29, a LCDR1 comprising SEQ ID NO: 33, a LCDR2 comprising SEQ IDNO: 34, and a LCDR3 comprising SEQ ID NO: 35. In some cases, theanti-TL1A antibody comprises a HCDR1 comprising SEQ ID NO: 30, a HCDR2comprising SEQ ID NO: 31, a HCDR3 comprising SEQ ID NO: 32, a LCDR1comprising SEQ ID NO: 33, a LCDR2 comprising SEQ ID NO: 34, and a LCDR3comprising SEQ ID NO: 35. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 36and a light chain (LC) variable domain comprising SEQ ID NO: 37. In somecases, the anti-TL1A antibody comprises a heavy chain comprising SEQ IDNO: 38. In some cases, the anti-TL1A antibody comprises a light chaincomprising SEQ ID NO: 39.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 40, a HCDR2 comprising SEQ ID NO: 41, a HCDR3 comprising SEQID NO: 42, a LCDR1 comprising SEQ ID NO: 43, a LCDR2 comprising SEQ IDNO: 44, and a LCDR3 comprising SEQ ID NO: 45. In some cases, theanti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 46 and a light chain (LC) variable domaincomprising SEQ ID NO: 47.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 48, a HCDR2 comprising SEQ ID NO: 49, a HCDR3 comprising SEQID NO: 50, a LCDR1 comprising SEQ ID NO: 51, a LCDR2 comprising SEQ IDNO: 52, and a LCDR3 comprising SEQ ID NO: 53. In some cases, theanti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 54 and a light chain (LC) variable domaincomprising SEQ ID NO: 55.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 56, a HCDR2 comprising SEQ ID NO: 58, a HCDR3 comprising SEQID NO: 59, a LCDR1 comprising SEQ ID NO: 61, a LCDR2 comprising SEQ IDNO: 63, and a LCDR3 comprising SEQ ID NO: 64. In some cases, theanti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 65 and a light chain (LC) variable domaincomprising SEQ ID NO: 69. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 65and a light chain (LC) variable domain comprising SEQ ID NO: 70. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 65 and a light chain (LC) variable domaincomprising SEQ ID NO: 71. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 65and a light chain (LC) variable domain comprising SEQ ID NO: 72.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 56, a HCDR2 comprising SEQ ID NO: 58, a HCDR3 comprising SEQID NO: 59, a LCDR1 comprising SEQ ID NO: 62, a LCDR2 comprising SEQ IDNO: 63, and a LCDR3 comprising SEQ ID NO: 64. In some cases, theanti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 65 and a light chain (LC) variable domaincomprising SEQ ID NO: 73. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 65and a light chain (LC) variable domain comprising SEQ ID NO: 74. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 65 and a light chain (LC) variable domaincomprising SEQ ID NO: 75. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 65and a light chain (LC) variable domain comprising SEQ ID NO: 76.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 56, a HCDR2 comprising SEQ ID NO: 58, a HCDR3 comprising SEQID NO: 59, a LCDR1 comprising SEQ ID NO: 61, a LCDR2 comprising SEQ IDNO: 63, and a LCDR3 comprising SEQ ID NO: 64. In some cases, theanti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 66 and a light chain (LC) variable domaincomprising SEQ ID NO: 69. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 66and a light chain (LC) variable domain comprising SEQ ID NO: 70. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 66 and a light chain (LC) variable domaincomprising SEQ ID NO: 71. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 66and a light chain (LC) variable domain comprising SEQ ID NO: 72.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 56, a HCDR2 comprising SEQ ID NO: 58, a HCDR3 comprising SEQID NO: 59, a LCDR1 comprising SEQ ID NO: 62, a LCDR2 comprising SEQ IDNO: 63, and a LCDR3 comprising SEQ ID NO: 64. In some cases, theanti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 66 and a light chain (LC) variable domaincomprising SEQ ID NO: 73. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 66and a light chain (LC) variable domain comprising SEQ ID NO: 74. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 66 and a light chain (LC) variable domaincomprising SEQ ID NO: 75. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 66and a light chain (LC) variable domain comprising SEQ ID NO: 76.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 57, a HCDR2 comprising SEQ ID NO: 58, a HCDR3 comprising SEQID NO: 60, a LCDR1 comprising SEQ ID NO: 61, a LCDR2 comprising SEQ IDNO: 63, and a LCDR3 comprising SEQ ID NO: 64. In some cases, theanti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 67 and a light chain (LC) variable domaincomprising SEQ ID NO: 69. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 67and a light chain (LC) variable domain comprising SEQ ID NO: 70. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 67 and a light chain (LC) variable domaincomprising SEQ ID NO: 71. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 67and a light chain (LC) variable domain comprising SEQ ID NO: 72. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 67 and a light chain (LC) variable domaincomprising SEQ ID NO: 73. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 67and a light chain (LC) variable domain comprising SEQ ID NO: 74. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 67 and a light chain (LC) variable domaincomprising SEQ ID NO: 75. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 67and a light chain (LC) variable domain comprising SEQ ID NO: 76.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 57, a HCDR2 comprising SEQ ID NO: 58, a HCDR3 comprising SEQID NO: 60, a LCDR1 comprising SEQ ID NO: 62, a LCDR2 comprising SEQ IDNO: 63, and a LCDR3 comprising SEQ ID NO: 64. In some cases, theanti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 68 and a light chain (LC) variable domaincomprising SEQ ID NO: 73. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 68and a light chain (LC) variable domain comprising SEQ ID NO: 74. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 68 and a light chain (LC) variable domaincomprising SEQ ID NO: 75. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 68and a light chain (LC) variable domain comprising SEQ ID NO: 76. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 68 and a light chain (LC) variable domaincomprising SEQ ID NO: 69. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 68and a light chain (LC) variable domain comprising SEQ ID NO: 70. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 68 and a light chain (LC) variable domaincomprising SEQ ID NO: 71. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 68and a light chain (LC) variable domain comprising SEQ ID NO: 72.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 77, a HCDR2 comprising SEQ ID NO: 78, a HCDR3 comprising SEQID NO: 79, a LCDR1 comprising SEQ ID NO: 80, a LCDR2 comprising SEQ IDNO: 81, and a LCDR3 comprising SEQ ID NO: 82. In some cases, theanti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 83 and a light chain (LC) variable domaincomprising SEQ ID NO: 88. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 83and a light chain (LC) variable domain comprising SEQ ID NO: 89. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 83 and a light chain (LC) variable domaincomprising SEQ ID NO: 90. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 83and a light chain (LC) variable domain comprising SEQ ID NO: 91. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 84 and a light chain (LC) variable domaincomprising SEQ ID NO: 88. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 84and a light chain (LC) variable domain comprising SEQ ID NO: 89. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 84 and a light chain (LC) variable domaincomprising SEQ ID NO: 90. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 84and a light chain (LC) variable domain comprising SEQ ID NO: 91. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 85 and a light chain (LC) variable domaincomprising SEQ ID NO: 88. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 85and a light chain (LC) variable domain comprising SEQ ID NO: 89. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 85 and a light chain (LC) variable domaincomprising SEQ ID NO: 90. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 85and a light chain (LC) variable domain comprising SEQ ID NO: 91. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 86 and a light chain (LC) variable domaincomprising SEQ ID NO: 88. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 86and a light chain (LC) variable domain comprising SEQ ID NO: 89. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 86 and a light chain (LC) variable domaincomprising SEQ ID NO: 90. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 86and a light chain (LC) variable domain comprising SEQ ID NO: 91. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 87 and a light chain (LC) variable domaincomprising SEQ ID NO: 88. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 87and a light chain (LC) variable domain comprising SEQ ID NO: 89. In somecases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 87 and a light chain (LC) variable domaincomprising SEQ ID NO: 90. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 87and a light chain (LC) variable domain comprising SEQ ID NO: 91.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 92, a HCDR2 comprising SEQ ID NO: 93, a HCDR3 comprising SEQID NO: 94, a LCDR1 comprising SEQ ID NO: 95, a LCDR2 comprising SEQ IDNO: 96, and a LCDR3 comprising SEQ ID NO: 97. In some cases, theanti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 98 and a light chain (LC) variable domaincomprising SEQ ID NO: 99. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 100and a light chain (LC) variable domain comprising SEQ ID NO: 101. Insome cases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 102 and a light chain (LC) variable domaincomprising SEQ ID NO: 103. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 104and a light chain (LC) variable domain comprising SEQ ID NO: 105. Insome cases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 106 and a light chain (LC) variable domaincomprising SEQ ID NO: 107. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 108and a light chain (LC) variable domain comprising SEQ ID NO: 109. Insome cases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 110 and a light chain (LC) variable domaincomprising SEQ ID NO: 111. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 112and a light chain (LC) variable domain comprising SEQ ID NO: 113. Insome cases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 114 and a light chain (LC) variable domaincomprising SEQ ID NO: 115. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 116and a light chain (LC) variable domain comprising SEQ ID NO: 117. Insome cases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 118 and a light chain (LC) variable domaincomprising SEQ ID NO: 119. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 120and a light chain (LC) variable domain comprising SEQ ID NO: 121.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 122, a HCDR2 comprising SEQ ID NO: 123, a HCDR3 comprisingSEQ ID NO: 124, a LCDR1 comprising SEQ ID NO: 125, a LCDR2 comprisingSEQ ID NO: 126, and a LCDR3 comprising SEQ ID NO: 127. In some cases,the anti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 128 and a light chain (LC) variable domaincomprising SEQ ID NO: 129.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 130, a HCDR2 comprising SEQ ID NO: 131, a HCDR3 comprisingSEQ ID NO: 132, a LCDR1 comprising SEQ ID NO: 133, a LCDR2 comprisingSEQ ID NO: 134, and a LCDR3 comprising SEQ ID NO: 135. In some cases,the anti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 136 and a light chain (LC) variable domaincomprising SEQ ID NO: 137.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 140, a HCDR2 comprising SEQ ID NO: 141, a HCDR3 comprisingSEQ ID NO: 142, a LCDR1 comprising SEQ ID NO: 143, a LCDR2 comprisingSEQ ID NO: 144, and a LCDR3 comprising SEQ ID NO: 145. In some cases,the anti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 138 and a light chain (LC) variable domaincomprising SEQ ID NO: 139. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 146and a light chain (LC) variable domain comprising SEQ ID NO: 147. Insome cases, the anti-TL1A antibody comprises a heavy chain (HC) variabledomain comprising SEQ ID NO: 148 and a light chain (LC) variable domaincomprising SEQ ID NO: 149. In some cases, the anti-TL1A antibodycomprises a heavy chain (HC) variable domain comprising SEQ ID NO: 150and a light chain (LC) variable domain comprising SEQ ID NO: 151.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 170, a HCDR2 comprising SEQ ID NO: 171, a HCDR3 comprisingSEQ ID NO: 172, a LCDR1 comprising SEQ ID NO: 173, a LCDR2 comprisingSEQ ID NO: 174, and a LCDR3 comprising SEQ ID NO: 175. In some cases,the anti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 176 and a light chain (LC) variable domaincomprising SEQ ID NO: 177.

In some embodiments, the anti-TL1A antibody comprises a HCDR1 comprisingSEQ ID NO: 178, a HCDR2 comprising SEQ ID NO: 179, a HCDR3 comprisingSEQ ID NO: 180, a LCDR1 comprising SEQ ID NO: 181, a LCDR2 comprisingSEQ ID NO: 182, and a LCDR3 comprising SEQ ID NO: 183. In some cases,the anti-TL1A antibody comprises a heavy chain (HC) variable domaincomprising SEQ ID NO: 184 and a light chain (LC) variable domaincomprising SEQ ID NO: 185.

In some embodiments, the anti-TL1A antibody is A100. In someembodiments, the anti-TL1A antibody is A101. In some embodiments, theanti-TL1A antibody is A102. In some embodiments, the anti-TL1A antibodyis A103. In some embodiments, the anti-TL1A antibody is A104. In someembodiments, the anti-TL1A antibody is A105. In some embodiments, theanti-TL1A antibody is A106. In some embodiments, the anti-TL1A antibodyis A107. In some embodiments, the anti-TL1A antibody is A108. In someembodiments, the anti-TL1A antibody is A109. In some embodiments, theanti-TL1A antibody is A110. In some embodiments, the anti-TL1A antibodyis Alll. In some embodiments, the anti-TL1A antibody is A112. In someembodiments, the anti-TL1A antibody is A113. In some embodiments, theanti-TL1A antibody is A114. In some embodiments, the anti-TL1A antibodyis A115. In some embodiments, the anti-TL1A antibody is A116. In someembodiments, the anti-TL1A antibody is A117. In some embodiments, theanti-TL1A antibody is A118. In some embodiments, the anti-TL1A antibodyis A119. In some embodiments, the anti-TL1A antibody is A120. In someembodiments, the anti-TL1A antibody is A121. In some embodiments, theanti-TL1A antibody is A122. In some embodiments, the anti-TL1A antibodyis A123. In some embodiments, the anti-TL1A antibody is A124. In someembodiments, the anti-TL1A antibody is A125. In some embodiments, theanti-TL1A antibody is A126. In some embodiments, the anti-TL1A antibodyis A127. In some embodiments, the anti-TL1A antibody is A128. In someembodiments, the anti-TL1A antibody is A129. In some embodiments, theanti-TL1A antibody is A130. In some embodiments, the anti-TL1A antibodyis A131. In some embodiments, the anti-TL1A antibody is A132. In someembodiments, the anti-TL1A antibody is A133. In some embodiments, theanti-TL1A antibody is A134. In some embodiments, the anti-TL1A antibodyis A135. In some embodiments, the anti-TL1A antibody is A136. In someembodiments, the anti-TL1A antibody is A137. In some embodiments, theanti-TL1A antibody is A138. In some embodiments, the anti-TL1A antibodyis A139. In some embodiments, the anti-TL1A antibody is A140. In someembodiments, the anti-TL1A antibody is A141. In some embodiments, theanti-TL1A antibody is A142. In some embodiments, the anti-TL1A antibodyis A143. In some embodiments, the anti-TL1A antibody is A144. In someembodiments, the anti-TL1A antibody is A145. In some embodiments, theanti-TL1A antibody is A146. In some embodiments, the anti-TL1A antibodyis A147. In some embodiments, the anti-TL1A antibody is A148. In someembodiments, the anti-TL1A antibody is A149. In some embodiments, theanti-TL1A antibody is A150. In some embodiments, the anti-TL1A antibodyis A151. In some embodiments, the anti-TL1A antibody is A152. In someembodiments, the anti-TL1A antibody is A153. In some embodiments, theanti-TL1A antibody is A154. In some embodiments, the anti-TL1A antibodyis A155. In some embodiments, the anti-TL1A antibody is A156. In someembodiments, the anti-TL1A antibody is A157. In some embodiments, theanti-TL1A antibody is A158. In some embodiments, the anti-TL1A antibodyis A159. In some embodiments, the anti-TL1A antibody is A160. In someembodiments, the anti-TL1A antibody is A161. In some embodiments, theanti-TL1A antibody is A162. In some embodiments, the anti-TL1A antibodyis A163. In some embodiments, the anti-TL1A antibody is A164. In someembodiments, the anti-TL1A antibody is A165. In some embodiments, theanti-TL1A antibody is A166. In some embodiments, the anti-TL1A antibodyis A167. In some embodiments, the anti-TL1A antibody is A168. In someembodiments, the anti-TL1A antibody is A169. In some embodiments, theanti-TL1A antibody is A170. In some embodiments, the anti-TL1A antibodyis A171. In some embodiments, the anti-TL1A antibody is A172. In someembodiments, the anti-TL1A antibody is A173. In some embodiments, theanti-TL1A antibody is A174. In some embodiments, the anti-TL1A antibodyis A175. In some embodiments, the anti-TL1A antibody is A174. In someembodiments, the anti-TL1A antibody is A176. In some embodiments, theanti-TL1A antibody is A174. In some embodiments, the anti-TL1A antibodyis A177.

In some embodiments, the anti-DR3 is A178. In some embodiments, theanti-DR3 is A179. In some embodiments, the anti-DR3 is A180. In someembodiments, the anti-DR3 is A181. In some embodiments, the anti-DR3 isA182. In some embodiments, the anti-DR3 is A183. In some embodiments,the anti-DR3 is A184. In some embodiments, the anti-DR3 is A185. In someembodiments, the anti-DR3 is A186. In some embodiments, the anti-DR3 isA187. In some embodiments, the anti-DR3 is A188. In some embodiments,the anti-DR3 is A189. In some embodiments, the anti-DR3 is A190. In someembodiments, the anti-DR3 is A191. In some embodiments, the anti-DR3 isA192. In some embodiments, the anti-DR3 is A193. In some embodiments,the anti-DR3 is A194. In some embodiments, the anti-DR3 is A195. In someembodiments, the anti-DR3 is A196. In some embodiments, the anti-DR3 isA197. In some embodiments, the anti-DR3 is A198. In some embodiments,the anti-DR3 is A199. In some embodiments, the anti-DR3 is A200. In someembodiments, the anti-DR3 is A201. In some embodiments, the anti-DR3 isA202. In some embodiments, the anti-DR3 is A203. In some embodiments,the anti-DR3 is A204. In some embodiments, the anti-DR3 is A205. In someembodiments, the anti-DR3 is A206. In some embodiments, the anti-DR3 isA207. In some embodiments, the anti-DR3 is A208. In some embodiments,the anti-DR3 is A209. In some embodiments, the anti-DR3 is A210. In someembodiments, the anti-DR3 is A211. In some embodiments, the anti-DR3 isA212. In some embodiments, the anti-DR3 is A213. In some embodiments,the anti-DR3 is A214. In some embodiments, the anti-DR3 is A215. In someembodiments, the anti-DR3 is A216. In some embodiments, the anti-DR3 isA217. In some embodiments, the anti-DR3 is A218. In some embodiments,the anti-DR3 is A219. In some embodiments, the anti-DR3 is A220. In someembodiments, the anti-DR3 is A221. In some embodiments, the anti-DR3 isA222. In some embodiments, the anti-DR3 is A223. In some embodiments,the anti-DR3 is A224. In some embodiments, the anti-DR3 is A225. In someembodiments, the anti-DR3 is A226. In some embodiments, the anti-DR3 isA227. In some embodiments, the anti-DR3 is A228. In some embodiments,the anti-DR3 is A229. In some embodiments, the anti-DR3 is A230. In someembodiments, the anti-DR3 is A231. In some embodiments, the anti-DR3 isA232. In some embodiments, the anti-DR3 is A233. In some embodiments,the anti-DR3 is A234. In some embodiments, the anti-DR3 is A235. In someembodiments, the anti-DR3 is A236. In some embodiments, the anti-DR3 isA237. In some embodiments, the anti-DR3 is A238. In some embodiments,the anti-DR3 is A239. In some embodiments, the anti-DR3 is A240. In someembodiments, the anti-DR3 is A241. In some embodiments, the anti-DR3 isA242.

In some cases, the anti-TL1A antibody binds to at least one or more ofthe same residues of human TL1A as an antibody described herein. Forexample, the anti-TL1A antibody binds to at least one or more of thesame residues of human TL1A as an antibody selected from A100-A177. Insome cases, the anti-TL1A antibody binds to the same epitope of humanTL1A as an antibody selected from A100-A177. In some cases, theanti-TL1A antibody binds to the same region of human TL1A as an antibodyselected from A100-A177. Non-limiting methods for determining whether ananti-TL1A antibody binds to the same region of a reference antibody areknown in the art. An exemplary method comprises a competition assay. Forinstance, the method comprises determining whether a reference antibodycan compete with binding between the reference antibody and the TL1Aprotein or portion thereof, or determining whether the referenceantibody can compete with binding between the reference antibody and theTL1A protein or portion thereof. Exemplary methods include use ofsurface plasmon resonance to evaluate whether an anti-TL1A antibody cancompete with the binding between TL1A and another anti-TL1A antibody. Insome cases, surface plasmon resonance is utilized in the competitionassay.

In some cases, the anti-CD30L antibody binds to at least one or more ofthe same residues of human CD30L as an antibody described herein. Forexample, the anti-CD30L antibody binds to at least one or more of thesame residues of human CD30L as an antibody selected from A178-A242. Insome cases, the anti-CD30L antibody binds to the same epitope of humanCD30L as an antibody selected from A178-A242. In some cases, theanti-CD30L antibody binds to the same region of human CD30L as anantibody selected from A178-A242.

TABLE 1 Non-Limiting Examples of anti-TL1A or anti-DR3 Antibodies andPortions Thereof SEQ ID Sequence   3 GFTFSTYG   4 ISGTGRTT   5TKERGDYYYG VFDY   6 QTISSW   7 AAS   8 QQYHRSWT   9EVQLLESGGG LVQPGKSLRL SCAVSGFTFS TYGMNWVRQA PGKGLEWVSSISGTGRTTYH ADSVQGRFTV SRDNSKNILY LQMNSLRADD TAVYFCTKERGDYYYGVFDY WGQGTLVTVS S  10DIQMTQSPST LSASVGDRVT ITCRASQTIS SWLAWYQQTP EKAPKLLIYAASNLQSGVPS RFSGSGSGTE FTLTISSLQP DDFATYYCQQ YHRSWTFGQG TKVEIT  11GFTFSSYW  12 IKEDGSEK  13 AREDYDSYYK YGMDV  14 QSILYSSNNK NY  15 WAS  16QQYYSTPFT  17 EVQLVESGGG LVQPGGSLRL SCAVSGFTFS SYWMSWVRQA PGKGLEWVANIKEDGSEKNY VDSVKGRFTL SSDNAKNSLY LQMNSLRAED TAVYYCAREDYDSYYKYGMD VWGQGTAVIV SS  18DIVMTQSPDS LAVSLGERAT INCKSSQSIL YSSNNKNYLA WYQQKPGQPPKLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVS VYYCQQYYST PFTFGPGTKV DIK 19 GGSFTGFY  20 INHRGNT  21 ASPFYDFWSG SDY  22 QSLVHSDGNT Y  23 KIS  24MQATQFPLT  25 QVQLQQWGAG LLKPSETLSL TCAVYGGSFT GFYWSWIRQP PGKGLEWIGEINHRGNTNYN PSLKSRVTMS VDTSKNQFSL NMISVTAADT AMYFCASPFYDFWSGSDYWG QGTLVTVSS  26DIMLTQTPLT SPVTLGQPAS ISCKSSQSLV HSDGNTYLSW LQQRPGQPPRLLFYKISNRF SGVPDRFSGS GAGTDFTLKI SRVEAEDVGV YYCMQATQFP LTFGGGTKVE IK  27GY(X1)F(X2)(X3)YGIS; X1 = P, S, D, Q, N; X2 = T, R; X3 = N, T, Y, H  28WIS(X1)YNG(X2)(X3)(X4)YA(X5)(X6)(X7)QG; X1 = T, P, S, A; X2 =N, G, V, K, A; X3 = T, K; X4 = H, N; X5 = Q, R; X6 = K, M; X7 = L, H  29ENYYGSG(X1)(X2)R GGMD(X3); X1 = S, A; X2 = Y, P;  X3 = V, A, G  30GYDFTYYGIS  31 WISTYNGNTH YARMLQG  32 ENYYGSGAYR GGMDV  33 RASQSVSSYL A 34 DASNRAT  35 QQRSNWPWT  36QVQLVQSGAE VKKPGASVKV SCKASGYDFT YYGISWVRQA PGQGLEWMGWISTYNGNTHY ARMLQGRVTM TTDTSTRTAY MELRSLRSDD TAVYYCARENYYGSGAYRGG MDVWGQGTTV TVSS  37EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPWTFGQ GTKVEIK  38QVQLVQSGAE VKKPGASVKV SCKASGYDFT YYGISWVRQA PGQGLEWMGWISTYNGNTHY ARMLQGRVTM TTDTSTRTAY MELRSLRSDD TAVYYCARENYYGSGAYRGG MDVWGQGTTV TVSSASTKGP SVFPLAPSSK STSGGTAALGCLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSLGTQTYICNVN HKPSNTKVDK KVEPKSCDKT HTCPPCPAPE AAGAPSVFLFPPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPREEQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQPREPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKTTPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPG  39EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPWTFGQGTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 40 SRSYYWG  41 SIYYNGRTYY NPSLKS  42 EDYGDYGAFD I  43 RASQGISSAL A  44DASSLES  45 QQFNSYPLT  46QLQLQESGPG LVKPSETLSL TCTVSGGSIS SRSYYWGWIR QPPGKGLEWIGSIYYNGRTY YNPSLKSRVT ISVDTSKNQF SLKLSSVTAA DTAVYYCAREDYGDYGAFDI WGQGTMVTVS S  47AIQLTQSPSS LSASVGDRVT ITCRASQGIS SALAWYQQKP GKAPKLLIYDASSLESGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGG GTKVEIK  48TSNMGVV  49 HILWDDREYSNPALKS  50 MSRNYYGSSYVMDY  51 SASSSVNYMH  52STSNLAS  53 HQWNNYGT  54QVTLKESGPALVKPTQTLTLTCTFSGFSLSTSNMGVVWIRQPPGKALEWLAHILWDDREYSNPALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARMSRNYYGSSYVMD YWGQGTLVTVSS  55DIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQWNNYGTFGQGTKVEIKR  56 LYGMN  57 NYGMN  58WINTYTGEPTYADDFKG  59 DTAMDYAMAY  60 DYGKYGDYYAMDY  61 KSSQNIVHSDGNTYLE 62 RSSQSIVHSNGNTYLD  63 KVSNRFS  64 FQGSHVPLT  65QVQLVQSGSELKKPGASVKVSCKASGYTFTLYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR DTAMDYAMAYWGQGTLVTVSS 66 QVQLVQSGSELKKPGASVKVSCKASGYTFTLYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCAR DTAMDYAMAYWGQGTLVTVSS 67 QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYGKYGDYYAMDYWGQGTLVTVSS  68QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDYGKYGDYYAMDYWGQGTLVTVSS  69DVVMTQSPLSLPVTLGQPASISCKSSQNIVHSDGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH VPLTFGGGTKVEIKR  70DVVMTQSPLSLPVTLGQPASISCKSSQNIVHSDGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH VPLTFGQGTKVEIKR  71DVVMTQTPLSLPVTPGEPASISCKSSQNIVHSDGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSH VPLTFGGGTKVEIKR  72DVVMTQTPLSLPVSLGDQASISCKSSQNIVHSDGNTYLEWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSH VPLTFGGGTKVEIKR  73DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLDWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH VPLTFGGGTKVEIKR  74DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLDWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH VPLTFGQGTKVEIKR  75DVVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLDWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSH VPLTFGGGTKVEIKR  76DVVMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLDWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCFQGSH VPLTFGGGTKLEIKR  77GYTFTSSWMH  78 IHPNSGGT  79 ARGDYYGYVS WFAY  80 QNINVL  81 KAS  82QQGQSYPYT  83 QVQLQQPGSV LVRPGASVKV SCKASGYTFT SSWMHWAKQR PGQGLEWIGEIHPNSGGTNY NEKFKGKATV DTSSSTAYVD LSSLTSEDSA VYYCARGDYYGYVSWFAYWG QGTLVTVSS  84QVQLVQSGAE VKKPGASVKV SCKASGYTFT SSWMHWARQA PGQGLEWIGEIHPNSGGTNY AQKFQGRATL TVDTSSSTAY MELSRLRSDD TAVYYCARGDYYGYVSWFAY WGQGTLVTVS S  85QVQLVQSGAE VKKPGASVKV SCKASGYTFT SSWMHWARQA PGQGLEWIGEIHPNSGGTNY AQKFQGRATM TVDTSISTAY MELSRLRSDD TAVYYCARGDYYGYVSWFAY WGQGTLVTVS S  86QVQLVQSGAE VKKPGASVKV SCKASGYTFT SSWMHWARQA PGQGLEWIGEIHPNSGGTNY AQKFQGRVTM TVDTSISTAY MELSRLRSDD TAVYYCARGDYYGYVSWFAY WGQGTLVTVS S  87QVQLVQSGAE VKKPGASVKV SCKASGYTFT SSWMHWARQA PGQGLEWMGEIHPNSGGTNY AQKFQGRVTM TVDTSISTAY MELSRLRSDD TAVYYCARGDYYGYVSWFAY WGQGTLVTVS S  88DIQMNQSPSS LSASLGDTIT ITCHASQNIN VLLSWYQQKP GNIPKLLIYKASNLHTGVPS RFSGSGSGTG FTFTISSLQP EDIATYYCQQ GQSYPYTFGG GTKLEIK  89DIQMTQSPSS LSASVGDRVT ITCQASQDIS NYLNWYQQKP GKAPKLLIYDASNLETGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ YDNLPYTFGQ GTKLEIK  90DIQMTQSPSS LSASVGDRVT ITCQASQNIN VLLNWYQQKP GKAPKLLIYKASNLHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ GQSYPYTFGQ GTKLEIK  91DIQMNQSPSS LSASVGDRVT ITCQASQNIN VLLSWYQQKP GKAPKLLIYKASNLHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQ GQSYPYTFGQ GTKLEIK  92GYTFTSYDIN  93 WLNPNSGXTG; X = N, Y  94 EVPETAAFEY  95TSSSSDIGA(X1) (X2)GV(X3); X1 = G, A; X2 = L, S, Q; X3 = H, L  96 GYYNRPS 97 QSXDGTLSAL; X = Y, W, F  98QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYDINWVRQA PGQGLEWMGWLNPNSGNTGY AQKFQGRVTM TADRSTSTAY MELSSLRSED TAVYYCAREVPETAAFEYWG QGTLVTVSS  99QSVLTQPPSV SGAPGQRVTI SCTSSSSDIG AXXGVXWYQQ LPGTAPKLLIEGYYNRPSGV PDRFSGSKSG TSASLTITGL LPEDEGDYYC QSXDGTLSAL FGGGTKLTVL G 100QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYDINWVRQA PGQGLEWMGWLNPNSGNTGY AQKFQGRVTM TADRSTSTAY MELSSLRSED TAVYYCAREVPETAAFEYWG QGTLVTVSS 101QSVLTQPPSV SGAPGQRVTI SCTSSSSDIG AGLGVHWYQQ LPGTAPKLLIEGYYNRPSGV PDRFSGSKSG TSASLTITGL LPEDEGDYYC QSWDGTLSAL FGGGTKLTVL G 102QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYDINWVRQA PGQGLEWMGWLNPNSGYTGY AQKFQGRVTM TADRSTSTAY MELSSLRSED TAVYYCAREVPETAAFEYWG QGTLVTVSS 103QSVLTQPPSV SGAPGQRVTI SCTSSSSDIG AGLGVHWYQQ LPGTAPKLLIEGYYNRPSGV PDRFSGSKSG TSASLTITGL LPEDEGDYYC QSYDGTLSAL FGGGTKLTVL G 104QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYDINWVRQA PGQGLEWMGWLNPNSGNTGY AQKFQGRVTM TADRSTSTAY MELSSLRSED TAVYYCAREVPETAAFEYWG QGTLVTVSS 105QSVLTQPPSV SGAPGQRVTI SCTSSSSDIG AALGVHWYQQ LPGTAPKLLIEGYYNRPSGV PDRFSGSKSG TSASLTITGL LPEDEGDYYC QSWDGTLSAL FGGGTKLTVL G 106QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYDINWVRQA PGQGLEWMGWLNPNSGNTGY AQKFQGRVTM TADRSTSTAY MELSSLRSED TAVYYCAREVPETAAFEYWG QGTLVTVSS 107QSVLTQPPSV SGAPGQRVTI SCTSSSSDIG AGSGVHWYQQ LPGTAPKLLIEGYYNRPSGV PDRFSGSKSG TSASLTITGL LPEDEGDYYC QSWDGTLSAL FGGGTKLTVL G 108QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYDINWVRQA PGQGLEWMGWLNPNSGNTGY AQKFQGRVTM TADRSTSTAY MELSSLRSED TAVYYCAREVPETAAFEYWG QGTLVTVSS 109QSVLTQPPSV SGAPGQRVTI SCTSSSSDIG AGQGVHWYQQ LPGTAPKLLIEGYYNRPSGV PDRFSGSKSG TSASLTITGL LPEDEGDYYC QSWDGTLSAL FGGGTKLTVL G 110QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYDINWVRQA PGQGLEWMGWLNPNSGNTGY AQKFQGRVTM TADRSTSTAY MELSSLRSED TAVYYCAREVPETAAFEYWG QGTLVTVSS 111QSVLTQPPSV SGAPGQRVTI SCTSSSSDIG AGLGVLWYQQ LPGTAPKLLIEGYYNRPSGV PDRFSGSKSG TSASLTITGL LPEDEGDYYC QSWDGTLSAL FGGGTKLTVL G 112QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYDINWVRQA PGQGLEWMGWLNPNSGYTGY AQKFQGRVTM TADRSTSTAY MELSSLRSED TAVYYCAREVPETAAFEYWG QGTLVTVSS 113QSVLTQPPSV SGAPGQRVTI SCTSSSSDIG AGLGVHWYQQ LPGTAPKLLIEGYYNRPSGV PDRFSGSKSG TSASLTITGL LPEDEGDYYC QSWDGTLSAL FGGGTKLTVL G 114QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYDINWVRQA PGQGLEWMGWLNPNSGYTGY AQKFQGRVTM TADRSTSTAY MELSSLRSED TAVYYCAREVPETAAFEYWG QGTLVTVSS 115QSVLTQPPSV SGAPGQRVTI SCTSSSSDIG AGSGVHWYQQ LPGTAPKLLIEGYYNRPSGV PDRFSGSKSG TSASLTITGL LPEDEGDYYC QSWDGTLSAL FGGGTKLTVL G 116QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYDINWVRQA PGQGLEWMGWLNPNSGYTGY AQKFQGRVTM TADRSTSTAY MELSSLRSED TAVYYCAREVPETAAFEYWG QGTLVTVSS 117QSVLTQPPSV SGAPGQRVTI SCTSSSSDIG AGQGVHWYQQ LPGTAPKLLIEGYYNRPSGV PDRFSGSKSG TSASLTITGL LPEDEGDYYC QSWDGTLSAL FGGGTKLTVL G 118QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYDINWVRQA PGQGLEWMGWLNPNSGYTGY AQKFQGRVTM TADRSTSTAY MELSSLRSED TAVYYCAREVPETAAFEYWG QGTLVTVSS 119QSVLTQPPSV SGAPGQRVTI SCTSSSSDIG AGLGVLWYQQ LPGTAPKLLIEGYYNRPSGV PDRFSGSKSG TSASLTITGL LPEDEGDYYC QSWDGTLSAL FGGGTKLTVL G 120QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYDINWVRQA PGQGLEWMGWLNPNSGYTGY AQKFQGRVTM TADRSTSTAY MELSSLRSED TAVYYCAREVPETAAFEYWG QGTLVTVSS 121QSVLTQPPSV SGAPGQRVTI SCTSSSSDIG AGLGVHWYQQ LPGTAPKLLIEGYYNRPSGV PDRFSGSKSG TSASLTITGL LPEDEGDYYC QSFDGTLSAL FGGGTKLTVL G 122SYFWS 123 YIYYSGNTKYNPSLKS 124 ETGSYYGFDY 125 RASQSINNYLN 126 AASSLQS127 QQSYSTPRT 128QVQLQESGPGLVKPSETLSLTCTVSGGSISSYFWSWIRQPPGKGLEWIGYIYYSGNTKYNPSLKSRVTISIDTSKNQFSLKLSSVTAADTAVYYCARETGSYYGFDYWGQGTLVTVS S 129DIQMTQSPSSLSASVGDRVTITCRASQSINNYLNWYQQRPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPGDFATYYCQQSYSTPRTFGQGTKLEIK 130 GYYWN 131EINHAGNTNYNPSLKS 132 GYCRSTTCYFDY 133 RASQSVRSSYLA 134 GASSRAT 135QQYGSSPT 136 QVQLQQWGAGLLKPSETLSLTCAVHGGSFSGYYWNWIRQPPGKGLEWIGEINHAGNTNYNPSLKSRVTISLDTSKNQFSLTLTSVTAADTAVYYCARGYCRSTTCYFDYWGQGTL VTVSS 137EIVLTQSPGTLSLSPGERATLSCRASQSVRSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPTFGQGTRLEIK 138EVQLQQSGAELVKPGASVKLSCTASGFDIQDTYMHWVKQRPEQGLEWIGRIDPASGHTKYDPKFQVKATITTDTSSNTAYLQLSSLTSEDTAVYYCSRSGGLPDVWGAGTTVTVS S 139QIVLSQSPAILSASPGEKVTMTCRASSSVSYMYWYQQKPGSSPKPWIYATSNLASGVPDRFSGSGSGTSYSLTISRVEAEDAATYYCQQWSGNPRTFGGGTKLEIK 140 GFDIQDTYMH 141RIDPASGHTKYDPKFQV 142 SGGLPDV 143 RASSSVSYMY 144 ATSNLAS 145 QQWSGNPRT146 QVQLVQSGAEVKKPGASVKLSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQVRVTMTTDTSTSTVYMELSSLRSEDTAVYYCSRSGGLPDVWGQGTTVT VSS 147EIVLTQSPGTLSLSPGERVTMSCRASSSVSYMYWYQQKPGQAPRPWIYATSNLASGVPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK 148QVQLVQSGAEVKKPGASVKLSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQVRVTMTRDTSTSTVYMELSSLRSEDTAVYYCSRSGGLPDVWGQGTTVT VSS 149EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK 150QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQVRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDVWGQGTTVT VSS 151EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGVPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK 152EVMLVESGGGLVKPGGSLKLSCAASGFTFTNYAMSWVRQTPEKRLEWVATITSGGSYIYYLDSVKGRFTISRDNAKSTLYLQMSSLRSEDTAIYNCARRKDGNYYYAMDYWGQG TSVTVSS 153EVMLVESGGGLVKPGGSLKLSCAMGFTFTNYAMSWVRQTPEKRLEWVATITSGGSYIYYLDSVKGRFTISRDNAKSTLYLQMSSLRSEDTAIYYCARRKDGNYYYAMDYWGQG TSVTVSS 154EVQLVESGGGLVKPGGSLRLSCAASGFTFTNYAMSWVRQAPGQRLEWVSTITSGGSYIYYLDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYNCARRKDGNYYYAMDYWGQ GTTVTVSS 155EVQLVESGGGLVKPGGSLRLSCAASGFTFTNYAMSWVRQAPGQRLEWVSTITSGGSYIYYLDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCARRKDGNYYYAMDYWGQ GTTVTVSS 156EVQLLESGGGLVQPGRSLRLSCAASGFTFTNYAMSWVRQAPGQRLEWLATITSGGSYIYYLDSVKGRFTISRDNSKSTLYLQMGSLRAEDMAVYNCARRKDGNYYYAMDYWGQ GTTVTVSS 157EVQLLESGGGLVQPGRSLRLSCAASGFTFTNYAMSWVRQAPGQRLEWLATITSGGSYIYYLDSVKGRFTISRDNSKSTLYLQMGSLRAEDMAVYYCARRKDGNYYYAMDYWGQ GTTVTVSS 158QVQLVESGGGLIQPGGSLRLSCAASGFTFTNYAMSWVRQARGQRLEWVSTITSGGSYIYYLDSVKGRFTISRDNSKSTLYMELSSLRSEDTAVYNCARRKDGNYYYAMDYWGQG TTVTVSS 159QVQLVESGGGLIQPGGSLRLSCAASGFTFTNYAMSWVRQARGQRLEWVSTITSGGSYIYYLDSVKGRFTISRDNSKSTLYMELSSLRSEDTAVYYCARRKDGNYYYAMDYWGQG TTVTVSS 160QVQLVQSGSELKKPGASVKVSCKASGFTFTNYAMSWVRQAPGKRLEWVSTITSGGSYIYYLDSVKGRFTISRENAKSTLYLQMNSLRTEDTALYNCARRKDGNYYYAMDYWGQ GTTVTVSS 161QVQLVQSGSELKKPGASVKVSCKASGFTFTNYAMSWVRQAPGKRLEWVATITSGGSYIYYLDSVKGRFTISRENAKSTLYLQMNSLRTEDTALYYCARRKDGNYYYAMDYWGQ GTTVTVSS 162EVQLLQSGAEVKKPGASVKVSCKASGFTFTNYAMSWVRQAPGQRLEWVATITSGGSYIYYLDSVKGRFTISRDNAKSTLHLQMNSLRAEDTAVYNCARRKDGNYYYAMDYWGQ GTTVTVSS 163EVQLLQSGAEVKKPGASVKVSCKASGFTFTNYAMSWVRQAPGQRLEWVATITSGGSYIYYLDSVKGRFTISRDNAKSTLHLQMNSLRAEDTAIYYCARRKDGNYYYAMDYWGQ GTTVTVSS 164EVMLLQSGAEVKKPGASVKVSCKASGFTFTNYAMSWVRQAPGQRLEWVATITSGGSYIYYLDSVKGRFTISRDNAKSTLHLQMNSLRAEDTAVYYCARRKDGNYYYAMDYWG QGTTVTVSS 165DIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFIHWYQQKAGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSYEDPWTFGGGTKLEIK 166DIVLTQSPATLSLSPGERATLSCRASESVDSYGNSFIHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTISSLEPEDFAVYYCQQSYEDPWTFGGGTKXEIK 167DIVLTQSPSSLSASVGDRVTITCRASESVDSYGNSFIHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTISSLQPEDFATYYCQQSYEDPWTFGGGTKXEIK 168DIVLTQSPDFQSVTPKEKVTITCRASESVDSYGNSFIHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTISSLEAEDAATYYCQQSYEDPWTFGGGTKXEIK 169DIVLTQTPLSLSVTPGQPASISCRASESVDSYGNSFIHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLKISRVEAEDVGVYYCQQSYEDPWTFGGGTKXEIK 170 TYGMS 171WMNTYSGVTTYADDFKG 172 EGYVFDDYYATDY 173 RSSQNIVHSDGNTYLE 174 KVSNRFS 175FQGSHVPLT 176 QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVKQAPGKGLKWMGWMNTYSGVTTYADDFKGRFAFSLETSASTAYMQIDNLKNEDTATYFCAREGYVFDDYYATDYW GQGTSVTVSS 177DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSDGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPLTFGAGTKLELK 178 KYDIN 179WIFPGDGRTDYNEKFKG 180 YGPAMDY 181 RSSQTIVHSNGDTYLD 182 KVSNRFS 183FQGSHVPYT 184 MGWSWVFLFLLSVTAGVHSQVHLQQSGPELVKPGASVKLSCKASGYTFTKYDINWVRQRPEQGLEWIGWIFPGDGRTDYNEKFKGKATLTTDKSSSTAYMEVSRLTSEDSAVYFCARYGPAMDYWGQGTSVTVA S 185MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGDTYLDWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK

TABLE 2 Non-Limiting Examples of anti-TL1A and anti-DR3 Antibodies HCVariable Domain LC Variable Domain Antibody Name (SEQ ID NO) (SEQ ID NO)A100 9 10 A101 17 18 A102 25 26 A103 36 37 A104 46 47 A105 54 55 A106 6569 A107 65 70 A108 65 71 A109 65 72 A110 65 73 A111 65 74 A112 65 75A113 65 76 A114 66 69 A115 66 70 A116 66 71 A117 66 72 A118 66 73 A11966 74 A120 66 75 A121 66 76 A122 67 69 A123 67 70 A124 67 71 A125 67 72A126 67 73 A127 67 74 A128 67 75 A129 67 76 A130 68 69 A131 68 70 A13268 71 A133 68 72 A134 68 73 A135 68 74 A136 68 75 A137 68 76 A138 83 88A139 83 89 A140 83 90 A141 83 91 A142 84 88 A143 84 89 A144 84 90 A14584 91 A146 85 88 A147 85 89 A148 85 90 A149 85 91 A150 86 88 A151 86 89A152 86 90 A153 86 91 A154 87 88 A155 87 89 A156 87 90 A157 87 91 A15898 99 A159 100 101 A160 102 103 A161 104 105 A162 106 107 A163 108 109A164 110 111 A165 112 113 A166 114 115 A167 116 117 A168 118 119 A169120 121 A170 128 129 A171 136 137 A172 138 139 A173 146 147 A174 148 149A175 150 151 A176 176 177 A177 184 185 A178 152 165 A179 152 166 A180152 167 A181 152 168 A182 152 169 A183 153 165 A184 153 166 A185 153 167A186 153 168 A187 153 169 A188 154 165 A189 154 166 A190 154 167 A191154 168 A192 154 169 A193 155 165 A194 155 166 A195 155 167 A196 155 168A197 155 169 A198 156 165 A199 156 166 A200 156 167 A201 156 168 A202156 169 A203 157 165 A204 157 166 A205 157 167 A206 157 168 A207 157 169A208 158 165 A209 158 166 A210 158 167 A211 158 168 A212 158 169 A213159 165 A214 159 166 A215 159 167 A216 159 168 A217 159 169 A218 160 165A219 160 166 A220 160 167 A221 160 168 A222 160 169 A223 161 165 A224161 166 A225 161 167 A226 161 168 A227 161 169 A228 162 165 A229 162 166A230 162 167 A231 162 168 A232 162 169 A233 163 165 A234 163 166 A235163 167 A236 163 168 A237 163 169 A238 164 165 A239 164 166 A240 164 167A241 164 168 A242 164 169

Methods disclosed herein may comprise administering any one of thedisclosed therapeutic agents alone. In other embodiments, methodsdisclosed herein may comprise administering any of the disclosedtherapeutic agents in combination with another therapeutic agentdisclosed herein, a nutritional-based therapy, a nature-based therapy, adiet-based therapy, or a combination thereof. A non-limiting example ofa nature-based therapy includes microbial-based treatments such asprobiotics.

Single Nucleotide Polymorphisms (SNPs) Associated with Non-Response toAnti-TNF Therapy

In one aspect, provided herein, a single nucleotide polymorphism (SNP)is detected in a biological sample obtained from the subject. The SNPmay be located at a gene locus involved in the mammalian innate andadaptive immune responses. In some embodiments, the gene locus isinvolved in the pathogenesis of IBD. In further embodiments, the genelocus is involved in autophagy and/or apoptosis. In some embodiments,the gene locus is in the major histocompatibility system, HLA. The genelocus may comprise TNFRSF1B, or HLA-DRB6, or a combination thereof.

In aspect, provided herein, a SNP at the TNFRSF1B gene locus comprisinga risk allele, “C” at nucleobase 256 within rs5745994 (SEQ ID NO:1), aportion of which is shown in Table 3, or any polymorphism in linkagedisequilibrium therewith is detected in a biological sample obtainedfrom the subject. In one embodiment, the SNP at the TNFRSF1B gene locuscomprises the risk allele, “C” at nucleobase 256 within SEQ ID NO. 1. ATNFRSF1B risk genotype may comprise a single copy of the risk allele,“C” at nucleobase 256 within rs5745994 (SEQ ID NO. 1). A TNFRSF1B riskgenotype may comprise two copies of the of the risk allele, “C” atnucleobase 256 within rs5745994 (SEQ ID NO. 1). In one aspect, providedherein, detection of the SNP at the TNFRSF1B gene locus is used topredict and/or diagnose non-response to anti-TNF therapy in a subject.In one aspect, provided herein, detection of the SNP at the TNFRSF1Bgene locus is used to treat the inflammatory disease or condition, orfibrostenotic and/or fibrotic disease, in a subject who is, or ispredicted to be, non-responsive to anti-TNF therapy. TNFRSF1B is a genethat encodes tumor necrosis factor receptor 2 (TNFR2), which is amembrane-bound receptor of TNF-α and a critical signaling protein in theimmune system. TNFR2, and nucleic acids encoding TNFR2, arecharacterized by NCBI Gene ID 7133.

In an aspect, provided herein, a SNP at the HLA-DRB6 gene locuscomprising a risk allele, “C” at nucleobase 501 within rs11757159 (SEQID NO: 2), a portion of which sequence is shown in of Table 3, or anySNP in linkage disequilibrium therewith, is detected in a biologicalsample obtained from the subject. In some embodiments, the SNP at theHLA-DRB6 gene locus comprises the risk allele, “C” at nucleobase 501within SEQ ID NO. 2. A HLA-DRB6 risk genotype may comprise a single copyof the risk allele, “C,” at nucleobase 501 within rs11757159 (SEQ ID NO.2). The HLA-DRB6 risk genotype may comprise two copies of the riskallele, “C,” at nucleobase 501 within rs11757159 (SEQ ID NO. 2). In oneaspect, provided herein, detection of the SNP at the HLA-DRB6 gene locusis used to predict and/or diagnose non-response to anti-TNF therapy. Inone aspect, provided herein, detection of the SNP at the HLA-DRB6 genelocus is used to treat the inflammatory disease or condition, orfibrostenotic and/or fibrotic disease, in a subject who is, or ispredicted to be, non-responsive to anti-TNF therapy. HLA-DRB6 is a genethat encodes a major histocompatibility complex, class II, DR beta 6(HLA-DRB6). HLA-DRB6, and nucleic acids encoding HLA-DRB6, arecharacterized by NCBI Gene ID 3128.

TABLE 3 SNPS Associated with Non-Response to anti-TNF  Therapy SEQ RiskID Gene Al- No. SNP  Locus lele Sequence 1 rs5745994 INFRSF1B CTGCTCCCGGG GGTCCTGGGA AGGCACAATG GTGACAGTGC TGCAGCTCTG CACTCCTGGAGGGTCACTCA GAGAC  [C/T] CGAGAGAGGAGGGCTCTGCG TCTGCTCCTC TGTCCAGGGCTGTAGCTTCT CTGGGTGCCT TTGCTTTTCT 2 rs11757159 HLA-DRB6 CTGGTTTCTCA TCTCAATGTT TGACAAGTTT GTTTCAGTTG TTATAGTCTG TTCTCAGTTTTTATGCACTG CCTTTTTGAA  [C/T] GTTAGGTTTA CTTTTTTAAT TGACAAGTAA AAATTGTATAGTATATTTAT GTTGTAGAGC ATGAAATTTT GATATATGCC

In some embodiments, provided is a method of detecting a genotype or SNPcomprising detecting the presence, absence, and/or quantity of a nucleicacid sequence, or portion thereof, selected from SEQ ID NOS: 1-2, or acombination thereof of Table 3. In some embodiments, provided is amethod of detecting a genotype or SNP comprising detecting the presence,absence, and/or quantity of a nucleic acid sequence, or portion thereof,selected from the reverse complement of SEQ ID NOS: 1-2, or acombination thereof of Table 3. In some cases, a portion of a nucleicacid sequence provided herein comprises at least about 10, 15, 20, 25,30, 35, 40, 45, or 50 contiguous nucleobases. In some cases, a portionof a nucleic acid sequence provided herein comprises between about 10and about 50 contiguous nucleobases, between about 10 and about 40contiguous nucleobases, between about 15 and about 50 contiguousnucleobases, between about 15 and about 40 contiguous nucleobases,between about 20 and about 50 contiguous nucleobases, and between about20 and about 40 contiguous nucleobases. In some cases, a portion of anucleic acid sequence provided herein comprises about 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50 contiguous nucleobases. In some cases, a portion of a nucleic acidsequence provided in Table 3 comprises a nucleobase within [brackets].In some cases, the portion comprises a “C” at nucleobase 501 within SEQID NO: 1. In some cases, the portion comprises a “C” at nucleobase 501within SEQ ID NO: 2. In some cases, e.g., when the reverse complement ofSEQ ID NO: 1 is detected, the portion comprises a “T” in the positionshown within brackets in Table 3. In some cases, e.g., when the reversecomplement of SEQ ID NO: 2 is detected, the portion comprises a “T” inthe position shown within brackets in Table 3.

In some embodiments, the method comprises determining the presence ofthe genotype or SNP in a sample obtained from a subject as determined bydetecting the presence or absence of: a nucleic acid sequence at leastor about 90% identical to SEQ ID NO: 1, a nucleic acid sequence at leastor about 90% identical to SEQ ID NO: 2, a nucleic acid sequence at leastor about 90% identical to a portion of SEQ ID NO: 1, a nucleic acidsequence at least or about 90% identical to a portion of SEQ ID NO: 2,or a combination thereof, in the genetic material. In some cases, if thesubject comprises the genotype or SNP, the subject is administered atherapeutic agent, as disclosed herein.

In some embodiments, the method comprises determining the presence ofthe genotype or SNP in a sample obtained from a subject as determined bydetecting the presence or absence of: a nucleic acid sequence at leastor about 95% identical to SEQ ID NO: 1, a nucleic acid sequence at leastor about 95% identical to SEQ ID NO: 2, a nucleic acid sequence at leastor about 95% identical to a portion of SEQ ID NO: 1, a nucleic acidsequence at least or about 95% identical to a portion of SEQ ID NO: 2,or a combination thereof, in the genetic material. In some cases, if thesubject comprises the genotype or SNP, the subject is administered atherapeutic agent, as disclosed herein.

In some embodiments, the method comprises determining the presence ofthe genotype or SNP in a sample obtained from a subject as determined bydetecting the presence or absence of: the reverse complement of anucleic acid sequence at least or about 90% identical to SEQ ID NO: 1,the reverse complement of a nucleic acid sequence at least or about 90%identical to SEQ ID NO: 2, the reverse complement of a nucleic acidsequence at least or about 90% identical to a portion of SEQ ID NO: 1,the reverse complement of a nucleic acid sequence at least or about 90%identical to a portion of SEQ ID NO: 2, or a combination thereof, in thegenetic material. In some cases, if the subject comprises the genotypeor SNP, the subject is administered a therapeutic agent, as disclosedherein.

In some embodiments, the method comprises determining the presence ofthe genotype or SNP in a sample obtained from a subject as determined bydetecting the presence or absence of: the reverse complement of anucleic acid sequence at least or about 95% identical to SEQ ID NO: 1,the reverse complement of a nucleic acid sequence at least or about 95%identical to SEQ ID NO: 2, the reverse complement of a nucleic acidsequence at least or about 95% identical to a portion of SEQ ID NO: 1,the reverse complement of a nucleic acid sequence at least or about 95%identical to a portion of SEQ ID NO: 2, or a combination thereof, in thegenetic material. In some cases, if the subject comprises the genotypeor SNP, the subject is administered a therapeutic agent, as disclosedherein.

Detecting TNFR2 Levels to Identify Non-Response to anti-TNF Therapy in aSubject

In one aspect, provided herein, a level of TNFR1 or TNFR2, or acombination thereof, is detected in a biological sample obtained from asubject with an inflammatory disease or condition, or fibrostenoticand/or fibrotic disease, In some embodiments, the level of TNFR1 orTNFR2, or combination thereof is lower as compared to an individual whodoes not express the TNRSF1B risk genotype. In some embodiments, thelevel of TNFR1 or TNFR2, or combination thereof is higher as compared toan individual who does not express the TNRSF1B risk genotype. In someembodiments, the level of TNFR1 or TNFR2, or combination thereof islower as compared to an individual who does not express the HLA-DRB6risk genotype. In some embodiments, the level of TNFR1 or TNFR2, orcombination thereof is higher as compared to an individual who does notexpress the HLA-DRB6 risk genotype. In one aspect, provided herein,detection of the level of TNFR1 or TNFR2 is used to predict and/ordiagnose non-response to anti-TNF therapy in a subject. In one aspect,provided herein, detection of the level of TNFR1 or TNFR2 is used totreat the inflammatory disease or condition, or fibrostenotic and/orfibrotic disease, in a subject who is, or is predicted to be,non-responsive to anti-TNF therapy. In some embodiments, a method ofdetecting a level of TNFR1 and/or TNFR2 comprises performing anenzyme-linked immunosorbent assay (ELISA) or other means known in theart for the detection of proteins, such as those described elsewhereherein.

TNFRSF1B is a gene that encodes tumor necrosis factor receptor 2(TNFR2), which is a membrane-bound receptor of TNF-α and a criticalsignaling protein in the immune system. TNFRSF1A is a gene that encodestumor necrosis factor receptor 1 (TNFR1), which is a membrane bound, orsoluble, receptor of TNFα, that also plays a role in the immune system.TNFR1 is expressed in almost all the cell types in human body, whereasTNFR2 is largely expressed in immune-related cells, such as lymphocytes(CD4 and CD8 cells), endothelia cells, and thymocytes. TNF-α binding toTNFR2 triggers the recruitment of the adapter proteins TNFreceptor-associated factor 1 (TRAF1) and TNF receptor-associated factor2 (TRAF2), precipitating activation of the downstream cascade of nuclearfactor-kappa B (NFkB) and c-Jun N-terminal kinase (JNK). This cascaderesults in proliferation of multiple immune-related cells, includingcytotoxic T cells, thymocytes, mononuclear cells as well as Treg cells,indicating the complex and crucial role of TNFR2 in human immuneresponse. TNFR2 has also been reported to mediate slan dendritic cell(slanDC) enhancement of NK-cell function, promote suppressive activitiesof myeloid-derived suppressor cells, and play a crucial auxiliary roleto TNFR1 in sensitizing macrophages for the activation of the p38mitogen-activated protein kinases (MAPK) and NFkB proinflammatorysignaling pathways.

Detecting ANCA Levels to Identify Non-Response to anti-TNF Therapy in aSubject

In one aspect, provided herein, a level of ANCA is detected in abiological sample obtained from a subject with an inflammatory diseaseor condition, or fibrostenotic and/or fibrotic disease. ANCA levels maycomprise a threshold level that serves as an independent indicator of asubject being, or being susceptible to, non-response to anti-TNFtherapy. In an embodiment provided herein, a first threshold ANCA levelis at or above about 100 ELISA units (EU), which is an independentindicator of the subject being, or being susceptible to, non-response toanti-TNF therapy. ANCA levels may comprise a second level that is lowerthan about 100 EU and above about 50 EU, which serves as an indicator ofa subject being, or being susceptible to, non-response to anti-TNFtherapy if either (i) a decrease in circulating TNFR2 levels, or (ii) apresence of the SNP at the TNFRSF1B gene locus, or a combinationthereof, is detected in the sample obtained from the subject. In anotherembodiment, the second threshold level of ANCA is at or above 50 EU. Insome embodiments, the level of ANCA is between 50 and 60 EU. In someembodiments, the ANCA level is between 60 and 70 EU. In someembodiments, the ANCA level is between 70 and 80 EU. In someembodiments, the ANCA level is between 80 and 90 EU. In someembodiments, the ANCA level is above 90 EU and below 100 EU. In oneaspect, provided herein, detection of ANCA levels, as disclosed herein,is used to predict and/or diagnose non-response to anti-TNF therapy in asubject. In one aspect, provided herein, detection of ANCA levels isused to treat the inflammatory disease or condition, or fibrostenoticand/or fibrotic disease, in a subject who is, or is predicted to be,non-responsive to anti-TNF therapy.

In some embodiments, the ANCA level is measured usingimmunohistochemistry. In some embodiments, the ANCA level is measuredusing an enzyme-linked immunosorbent assay (ELISA). The ELISA may be afixed-ELISA. The fixed-ELISA may be fixed granulocytes as disclosed, forexample, in Saxon et al., A distinct subset of antineutrophilcytoplasmic antibodies is associated with inflammatory bowel disease, J.Allergy Clin. Immuno. 86:2; 202-210 (August 1990).

Previous studies suggest that serum ANCA is a result of reactivity tolocal antigens. Previous studies have shown that the antigen forIBD-associated ANCA is nuclear and reactivity can be eliminated bypretreating neutrophils with DNase, which suggests that ANCA could be amarker for increased nuclear destruction within the mucosa involvingrelease of nuclear antigens potentially related to ongoing apoptoticactivity. Inhibition of mucosal inflammation by TNFα is driven byinduction of T-cell apoptosis. Apoptosis is dependent on a balance ofpro versus anti-apoptotic signal generating molecules that induce orprotect against apoptosis. Membrane TNF, present on macrophages,preferentially binds to TNFR2 as opposed to TNFR1 on activated CD4 Tcells. Downstream signaling of TNFR2 activates NFKB, which inducesintracellular molecules and soluble cytokines that protect the cell fromproapoptotic signals and raise the threshold for apoptosis in CD4 Tcells. The result is increasing numbers of activated mucosal T cells inpatients with IBD. Therapeutic antibodies to TNF bind transmembrane TNF,potentially preventing TNFR2 signaling that lowers the apoptoticthreshold and makes these cells more susceptible to ongoing apoptosis,resulting in decreased numbers of mucosal effector T cells. Thus,without being bound to any particular theory, high ANCA levels and lowerlevels of TNFR2 (reflected by lower circulating TNFR2 levels) suggestongoing mucosal apoptosis. Therefore, treating this population withanti-TNF will not further enhance apoptosis and a therapeutic thattargets different mechanisms, such as a therapeutic targeting TL1A,would be indicated.

Detecting the TNRSF1B Risk Genotype, Decreased TNFR2, and ANCA Level toIdentify Non-Response to Anti-TNF Therapy in Subjects with IBD

In one aspect, provided herein, detection of a TNFRSF1B risk genotype,an increased level of ANCA, and decreased circulating TNFR2, in abiological sample obtained from a subject with an inflammatory diseaseor condition, or fibrostenotic and/or fibrotic disease, is used toidentify or predict non-response to anti-TNF therapy in the subject. Inone aspect, provided herein, detection of a TNFRSF1B risk genotype, anincreased level of ANCA, and decreased circulating TNFR2, in abiological sample obtained from a subject with an inflammatory diseaseor condition, or fibrostenotic and/or fibrotic disease, is used to treatthe inflammatory disease or condition, or fibrostenotic and/or fibroticdisease. In aspect, provided herein, a SNP at the TNFRSF1B gene locuscomprising a risk allele, “C” at nucleobase 256 within rs5745994 (SEQ IDNO: 1), a portion of which sequence shown in of Table 3, or anypolymorphism in linkage disequilibrium therewith is detected in abiological sample obtained from the subject. In one embodiment, the SNPat the TNFRSF1B gene locus comprises the risk allele, “C” at nucleobase256 within SEQ ID NO. 1. A TNFRSF1B risk genotype may comprise a singlecopy of the risk allele, “C” at nucleobase 256 within rs5745994 (SEQ IDNO. 1). A TNFRSF1B risk genotype may comprise two copies of the of therisk allele, “C” at nucleobase 256 within rs5745994 (SEQ ID NO. 1). Insome embodiments, the TNFRSF1B risk genotype may be detected bycontacting the biological sample obtained from the subject with anucleic acid sequence capable of hybridizing to 10 nucleobases of SEQ IDNO. 1 spanning nucleobase 256 under standard hybridization conditions.In some embodiments, the standard hybridization conditions comprise anannealing temperature between about 30° C. and about 65° C. In anembodiment provided herein, a first threshold ANCA level is at or aboveabout 100 ELISA units (EU), which is an independent indicator that thesubject is, or is susceptible to non-response to anti-TNF therapy. ANCAlevels may comprise a level that is lower than about 100 EU and aboveabout 50 EU, which serves as an indicator that the subject is, or issusceptible to, non-response to anti-TNF therapy if either (i) adecrease in circulating TNFR2 levels, or (ii) a presence of the SNP atthe TNFRSF1B gene locus, or a combination thereof, is detected in thebiological sample obtained from the subject. In another embodiment, asecond threshold level of ANCA is at or above 50 EU. In someembodiments, the level of ANCA is between 50 and 60 EU. In someembodiments, the ANCA level is between 60 and 70 EU. In someembodiments, the ANCA level is between 70 and 80 EU. In someembodiments, the ANCA level is between 80 and 90 EU. In someembodiments, the ANCA level is above 90 EU and below 100 EU. In someembodiments, the ANCA level is measured using immunohistochemistry. Insome embodiments, the ANCA level is measured using an enzyme-linkedimmunosorbent assay (ELISA). The ELISA may be a fixed-ELISA. Thefixed-ELISA may be fixed with granulocytes as disclosed, for example, inSaxon et al., A distinct subset of antineutrophil cytoplasmic antibodiesis associated with inflammatory bowel disease, J. Allergy Clin. Immuno.86:2; 202-210 (August 1990). In some embodiments, the level of TNFR2 islower as compared to an individual who does not express the TNRSF1B riskgenotype. In some embodiments, the level of TNFR2 is measured usingimmunohistochemistry. In some embodiments, the level of TNFR2 ismeasured using an ELISA.

Methods of Selecting a Therapy

In an aspect, provided herein, are methods of selecting a therapy for asubject with inflammatory disease or condition, or fibrostenotic and/orfibrotic disease, comprising requesting the results of whether thesubject is non-responsive to anti-TNF therapy, wherein the results areobtained by (i) obtaining a biological sample from the subject, (ii)assaying the sample to determine the presence of a TNFRSF1B SNP, (iii)assaying the sample for a serological factor comprising ANCA;determining the subject as non-responsive to anti-TNF therapy based onthe presence of a TNFRSF1B SNP and an increase in the serological factorrelative to a reference value obtained from an individual who does notexpress the TNFRSF1B SNP; and selecting a non-anti-TNF therapy for thesubject. In some embodiments, the non-anti-TNF therapy comprises ananti-TL1A therapy. In some embodiments, the anti-TL1A therapy comprisesan anti-TL1A antibody. In some embodiments, the anti-TL1A therapycomprises a blocking TL1A antibody. In some embodiments, inflammatorydisease and/or condition comprises IBD. In some embodiments, the IBDcomprises Crohn's disease or ulcerative colitis. In some embodiments,the method further comprises assaying for TNFR2 levels. In otherembodiments, a decrease in TNFR2 levels as compared to an individual whodoes not express the TNFRSF1B SNP, is indicative of a subjectnon-responsive to anti-TNF therapy. In various embodiments, the methodfurther comprises administering the non-anti-TNF therapy, which includesbut is not limited, to those disclosed above. In other embodiments, themethod further comprises assaying the biological sample for the presenceof a HLA-DRB6 SNP. In various embodiments, the biological sample isassayed for the HLA-DRB6 SNP and a serological factor. In someembodiments, the HLA-DRB6 SNP variant is rs11757159 (SEQ ID NO: 2). Insome embodiments, the serological factor is ANCA. In some embodiments,the presence of the HLA-DRB6 SNP is associated with a decrease in ANCA.

Compositions and Kits

In an aspect, disclosed herein, are kits to identify a subject with IBD,CD and/or UC that is non-responsive to anti-TNF therapy and/or selectinga therapy for a subject with IBD, CD and/or UC in need of treatment. Thekit is useful for practicing the methods disclosed herein. The kit is anassemblage of materials or components, including at least one of theinventive compositions. Thus, in some embodiments the kit contains acomposition including primers and probes for detecting the proteinsand/or genes comprising TNFRSF1B, ANCA, TNFR2 and/or HLA-DRB6, asdescribed above.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. For example, some embodiments areconfigured for the purpose of assessing risk variants, protein level,and/or gene expression levels. In some embodiments, the kit isconfigured to detect a presence, or gene expression levels, of TNFRSF1Bin a sample. In yet other embodiments, the kit is configured to detectthe presence, or gene expression levels, of gene expression productsfrom the gene TNFRSF1B and/or HLA-DRB6 in a sample. Gene expressionproduct from a gene may be RNA and/or protein expressed from the gene.

Other embodiments are configured for the purpose of assessing proteinlevels. In some embodiments, the kit is configured to detect the proteinlevels of TNFR1, TNFR2, and/or ANCA in a sample. In some otherembodiments, the kit is configured to detect the protein levels of ANCA,TNFR2 and/or HLA-DRB6 in a sample. In one embodiment, the kit isconfigured to detect TNFRSF1B and/or HLA-DRB6 risk genotypes in asample.

In one embodiment, the kit is configured particularly for the purpose ofassessing mammalian subjects. In another embodiment, the kit isconfigured particularly for the purpose of assessing human subjects. Infurther embodiments, the kit is configured for veterinary applications,assessing subjects such as, but not limited to, farm animals, domesticanimals, and laboratory animals.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to identify a subject with IBD, CD and/or UC that isnon-responsive to anti-TNF therapy and/or select a therapy for, or treatusing the therapy, a subject with IBD, CD and/or UC in need oftreatment. In some embodiments, the therapy comprises an inhibitor ofTL1A activity or expression. In some embodiments, the inhibitor of TL1Aactivity or expression comprises an anti-TL1A antibody. Optionally, thekit also contains other useful components, such as, primers, diluents,buffers, pipetting or measuring tools or other useful paraphernalia aswill be readily recognized by those of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well-knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging materials employed in the kit are those customarilyutilized in gene and/or protein expression assays. As used herein, theterm “package” refers to a suitable solid matrix or material such asglass, plastic, paper, foil, and the like, capable of holding theindividual kit components. Thus, for example, a package can be a glassvial used to contain suitable quantities of an inventive compositioncontaining primers and probes for detection of proteins and/or genesselected from TNFRSF1B, ANCA, TNFR2 and/or HLA-DRB6. The packagingmaterial generally has an external label which indicates the contentsand/or purpose of the kit and/or its components.

An aspect, provided herein, are compositions comprising at least 10 butless than 50 contiguous nucleobase residues of SEQ ID NOs. 1 or 2,wherein the contiguous nucleobase residues comprise the nucleobase atposition 256 of SEQ ID No. 1, or position 501 at SEQ ID No. 2, andwherein the contiguous nucleobase residues are connected to a detectablemolecule. The detectable molecule may be any molecule suitable fornucleic acid detection. In some embodiments, the detectable molecule isa fluorophore. In some embodiments the contiguous nucleobase residuesare connected to a quencher. As a non-limiting example, the compositionsare a probe useful for detection of the presence or absence of a SNPdisclosed herein. An aspect, provided herein are kits comprising a probedescribed herein and a primer pair, each primer having a nucleic acidsequence hybridizable to 10 different contiguous nucleobases of SEQ IDNO: 1, wherein the primer pair is configured to amplify at least about30 nucleobases of SEQ ID NO: 1 in a standard amplification assay (e.g.,PCR), and wherein the at least 30 nucleobases comprise nucleobase 256 ofSEQ ID NO. 1. In another aspect, provided herein are kits comprising aprobe described herein and a primer pair, each primer having a nucleicacid sequence hybridizable to 10 different contiguous nucleobases of SEQID NO: 2, wherein the at least about 30 nucleobases comprise nucleobase501 of SEQ ID NO. 2. In some embodiments, methods are provided forcontacting DNA from a subject with the composition described herein, orusing the kit described herein under conditions configured to hybridizethe composition to the DNA if the DNA comprises a sequence complementaryto the composition. In further embodiments, provided herein are methodsof treating the subject with an inhibitor of TL1A activity orexpression, provided that the DNA from the subject comprises thesequence complementary to the composition. In some embodiments, the kitdisclosed herein comprises an ELISA. In some embodiments, the ELISA is afixed-ELISA. In some embodiments, the fixed-ELISA comprises purifiedphosphopeptidomannan derived from yeast configured to detect a level ofANCA, as disclosed herein.

Biological Samples, Sample Preparation and Genotype Detection

In an aspect, provided herein, the steps involve obtaining a biologicalsample from a subject. The biological sample may be obtained eitherthrough surgical biopsy or surgical resection. Alternatively, a samplecan be obtained through primary patient derived cell lines, or archivedpatient samples in the form of FFPE (Formalin fixed, paraffin embedded)samples, or fresh frozen samples. A sample may also comprise wholeblood, peripheral blood, plasma, serum, saliva, cheek swab, or otherbodily fluid or tissue. In various embodiments, the sample comprisestissue from the large and/or small intestine. In various otherembodiments, the large intestine sample comprises the cecum, colon (theascending colon, the transverse colon, the descending colon, and thesigmoid colon), rectum and/or the anal canal. In yet other embodiments,the small intestine sample comprises the duodenum, jejunum, and/or theileum. In some embodiments, the sample is a blood sample. In yet otherembodiments, the sample is serum. In yet other embodiments, two samplesare collected. In some embodiments, the samples are tissue and blood.

Nucleic acid or protein samples derived from the biological sample(i.e., tissue and/or cells) of a subject that can be used in the methodsdisclosed herein can be prepared by means well known in the art. Forexample, surgical procedures or needle biopsy aspiration can be used tocollect the biological samples from a subject. In some embodiments, itis important to enrich and/or purify the abnormal tissue and/or cellsamples from the normal tissue and/or cell samples. In otherembodiments, the abnormal tissue and/or cell samples can then bemicrodissected to reduce the amount of normal tissue contamination priorto extraction of genomic nucleic acid or pre-RNA for use in the methodsdisclosed herein. Such enrichment and/or purification can beaccomplished according to methods well-known in the art, such as needlemicrodissection, laser microdissection, fluorescence activated cellsorting, and immunological cell sorting.

Analysis of the nucleic acid and/or protein from an individual may beperformed using any of various techniques. In various embodiments,assaying gene expression products from the genes TNFRSF1B, TNFRSF1A,and/or HLA-DRB6 comprises northern blot, reverse transcription PCR,real-time PCR, serial analysis of gene expression (SAGE), DNAmicroarray, tiling array, RNA-Seq, or a combination thereof. In variousother embodiments, the levels of gene expression products from the genesTNFRSF1B, TNFRSF1A, and/or HLA-DRB6 are assayed. In various otherembodiments, the level of gene expression products from the geneTNFRSF1B are assayed. In various other embodiments, the levels of geneexpression products from the gene HLA-DRB6 are assayed.

In various other embodiments, determining the protein expression levelof ANCA, TNFR1 and/or TNFR2 can be accomplished by analyzing theproteins of a biological sample from the subject. In variousembodiments, methods and systems to detect ANCA, TNFR1 and/or TNFR2,include but are not limited to enzyme-linked immunosorbent assay(ELISA), immunohistochemistry, western blot, flow cytometry,fluorescence in situ hybridization (FISH), radioimmuno assays, andaffinity purification. In various embodiments, the assay is an ELISA,including but not limited to indirect ELISA, sandwich ELISA, competitiveELISA, multiple and portable ELISA, and fixed-ELISA. In variousembodiments the ELISA is a fixed neutrophil ELISA. In some embodiments,the ELISA comprises fixed granulocytes, as disclosed, for example, inSaxon et al., A distinct subset of antineutrophil cytoplasmic antibodiesis associated with inflammatory bowel disease, J. Allergy Clin. Immuno.86:2; 202-210 (August 1990).

The analysis of gene expression levels may involve amplification of anindividual's nucleic acid by the polymerase chain reaction. Use of thepolymerase chain reaction for the amplification of nucleic acids is wellknown in the art (see, for example, Mullis et al. (Eds.), The PolymeraseChain Reaction, Birkhauser, Boston, (1994)).

Methods of “quantitative” amplification may be used to detect theTNFRSF1 or HLA-DRB6 risk genotype. For example, quantitative PCRinvolves simultaneously co-amplifying a known quantity of a controlsequence using the same primers. This provides an internal standard thatmay be used to calibrate the PCR reaction. Detailed protocols forquantitative PCR are provided in Innis, et al. (1990) PCR Protocols, AGuide to Methods and Applications, Academic Press, Inc. N.Y.).Measurement of DNA copy number at microsatellite loci using quantitativePCR analysis is described in Ginzonger, et al. (2000) Cancer Research60:5405-5409. The known nucleic acid sequence for the genes issufficient to enable one of skill in the art to routinely select primersto amplify any portion of the gene. Fluorogenic quantitative PCR mayalso be used in the methods disclosed herein. In fluorogenicquantitative PCR, quantitation is based on amount of fluorescencesignals, e.g., TaqMan and sybr green.

Other suitable amplification methods include, but are not limited to,ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560,Landegren, et al. (1988) Science 241:1077, and Barringer et al. (1990)Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc.Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication(Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR,and linker adapter PCR, etc.

A DNA sample suitable for hybridization can be obtained, e.g., bypolymerase chain reaction (PCR) amplification of genomic DNA, fragmentsof genomic DNA, fragments of genomic DNA ligated to adaptor sequences orcloned sequences. Computer programs can be used in the design of primerswith the desired specificity and optimal amplification properties, suchas Oligo version 5.0 (National Biosciences). PCR methods are described,for example, in Innis et al., eds., 1990, PCR Protocols: A Guide toMethods And Applications, Academic Press Inc., San Diego, Calif. It willbe apparent to one skilled in the art that controlled robotic systemsare useful for isolating and amplifying nucleic acids and can be used.

A variety of methods can be used to determine the presence the TNFRSF1or HLA-DRB6 risk genotype. As an example, enzymatic amplification ofnucleic acid from an individual may be used to obtain nucleic acid forsubsequent analysis, as discussed above. The presence or absence of avariant allele or haplotype may also be determined directly from theindividual's nucleic acid without enzymatic amplification.

A TaqmanB allelic discrimination assay available from Applied Biosystemsmay be useful for detecting the TNFRSF1 or HLA-DRB6 risk genotype. In aTaqmanB allelic discrimination assay, a specific, fluorescent,dye-labeled probe for each allele is constructed. The probes containdifferent fluorescent reporter dyes such as FAM and VIC™ todifferentiate the amplification of each allele. In addition, each probehas a quencher dye at one end which quenches fluorescence byfluorescence resonant energy transfer (FRET). During PCR, each probeanneals specifically to complementary sequences in the nucleic acid fromthe individual. The 5′ nuclease activity of Taq polymerase is used tocleave only probe that hybridize to the allele. Cleavage separates thereporter dye from the quencher dye, resulting in increased fluorescenceby the reporter dye. Thus, the fluorescence signal generated by PCRamplification indicates which alleles are present in the sample.Mismatches between a probe and allele reduce the efficiency of bothprobe hybridization and cleavage by Taq polymerase, resulting in littleto no fluorescent signal. Improved specificity in allelic discriminationassays can be achieved by conjugating a DNA minor grove binder (MGB)group to a DNA probe as described, for example, in Kutyavin et al.,“3′-minor groove binder-DNA probes increase sequence specificity at PCRextension temperature, “Nucleic Acids Research 28:655-661 (2000)). Minorgrove binders include, but are not limited to, compounds such asdihydrocyclopyrroloindole tripeptide (DPI).

Sequence analysis also may also be useful for detecting the TNFRSF1 orHLA-DRB6 risk genotype. In some embodiments, DNA is fully or partiallyisolated and/or purified from other cellular material prior to or duringDNA sequencing of the biological sample. In some instances, DNA isisolated and/or purified from other cellular materials by standard DNApurification techniques including, but not limited to, organicextraction (phenol, chloroform, and/or isoamyl alcohol), cesium chloridedensity gradients, anion-exchange methods, and selective adsorption tosilica. Commercial kits available to at least partially isolate and/orpurify DNA from cellular material include, but are not limited to,QIAamp (Qiagen), DNeasy (Qiagen), Quick-gDNA™ (Zymo Research), ZR-96Quick-gDNA (Zymo Research), Xpedition™ (Zymo Research), DNAzol® (LifeTechnologies), ChargeSwitch® gDNA Mini Tissue Kit (Life Technologies),PureLink® (Life Technologies), GeneCatcher™ (Life Technologies),ChargeSwitch® Forensic DNA Purification Kit (Life Technologies),ReliaPrep™ (Promega), and Wizard® (Promega). In some instances, DNA issequenced by traditional sequencing methods (see, A. M. Maxam and W.Gilbert, Proc. Natl. Acad. Sci. USA 74:560 (1977); Sanger et al., Proc.Natl. Acad. Sci. USA 74:5463 (1977)), next-generation sequencing methods(see, Mardis E R, Annu. Rev. Genomics Hum. Genet. USA 9:387 (2008)), oradditional methods known to one of skill in the art.

Restriction fragment length polymorphism (RFLP) analysis may also beuseful for detecting the TNFRSF1 or HLA-DRB6 risk genotype (Jarcho etal. in Dracopoli et al., Current Protocols in Human Genetics pages2.7.1-2.7.5, John Wiley & Sons, New York; Innis et al., (Ed.), PCRProtocols, San Diego: Academic Press, Inc. (1990)). As used herein,restriction fragment length polymorphism analysis is any method fordistinguishing genetic polymorphisms using a restriction enzyme, whichis an endonuclease that catalyzes the degradation of nucleic acid andrecognizes a specific base sequence, generally a palindrome or invertedrepeat. One skilled in the art understands that the use of RFLP analysisdepends upon an enzyme that can differentiate two alleles at apolymorphic site.

Allele specific oligonucleotide hybridization may also be used to detecta disease-predisposing allele. Allele-specific oligonucleotidehybridization is based on the use of a labeled oligonucleotide probehaving a sequence perfectly complementary, for example, to the sequenceencompassing a disease-predisposing allele. Under appropriateconditions, the allele-specific probe hybridizes to a nucleic acidcontaining the disease-predisposing allele but does not hybridize to theone or more other alleles, which have one or more nucleotide mismatchesas compared to the probe. If desired, a second allele-specificoligonucleotide probe that matches an alternate allele also can be used.Similarly, the technique of allele-specific oligonucleotideamplification can be used to selectively amplify, for example, adisease-predisposing allele by using an allele-specific oligonucleotideprimer that is perfectly complementary to the nucleotide sequence of thedisease-predisposing allele but which has one or more mismatches ascompared to other alleles (Mullis et al., supra, (1994)). One skilled inthe art understands that the one or more nucleotide mismatches thatdistinguish between the disease-predisposing allele and one or moreother alleles are preferably located in the center of an allele-specificoligonucleotide primer to be used in allele-specific oligonucleotidehybridization. In contrast, an allele-specific oligonucleotide primer tobe used in PCR amplification preferably contains the one or morenucleotide mismatches that distinguish between the disease-associatedand other alleles at the 3′ end of the primer.

A heteroduplex mobility assay (HMA) is another assay that may be used todetect the TNFRSF1 or HLA-DRB6 risk genotype. HMA is useful fordetecting the presence of a polymorphic sequence since a DNA duplexcarrying a mismatch has reduced mobility in a polyacrylamide gelcompared to the mobility of a perfectly base-paired duplex (Delwart etal., Science 262:1257-1261 (1993); White et al., Genomics 12:301-306(1992)).

The technique of single strand conformational, polymorphism (SSCP) alsomay be used to detect the presence of the TNFRSF1 or HLA-DRB6 riskgenotype (see Hayashi, K., Methods Applic. 1:34-38 (1991)). Thistechnique can be used to detect mutations based on differences in thesecondary structure of single-strand DNA that produce an alteredelectrophoretic mobility upon non-denaturing gel electrophoresis.Polymorphic fragments are detected by comparison of the electrophoreticpattern of the test fragment to corresponding standard fragmentscontaining known alleles.

Denaturing gradient gel electrophoresis (DGGE) also may be used todetect the TNFRSF1 or HLA-DRB6 risk genotype. In DGGE, double-strandedDNA is electrophoresed in a gel containing an increasing concentrationof denaturant; double-stranded fragments made up of mismatched alleleshave segments that melt more rapidly, causing such fragments to migratedifferently as compared to perfectly complementary sequences (Sheffieldet al., “Identifying DNA Polymorphisms by Denaturing Gradient GelElectrophoresis” in Innis et al., supra, 1990).

Other molecular methods useful for determining the presence of theTNFRSF1 or HLA-DRB6 risk genotype. Other well-known approaches fordetermining the presence of the TNFRSF1 or HLA-DRB6 risk genotypeinclude automated sequencing and RNAase mismatch techniques (Winter etal., Proc. Natl. Acad. Sci. 82:7575-7579 (1985)). Furthermore, oneskilled in the art understands that, where the presence or absence ofmultiple alleles or haplotype(s) is to be determined, individual allelescan be detected by any combination of molecular methods. See, ingeneral, Birren et al. (Eds.) Genome Analysis: A Laboratory ManualVolume 1 (Analyzing DNA) New York, Cold Spring Harbor Laboratory Press(1997). In addition, one skilled in the art understands that multiplealleles can be detected in individual reactions or in a single reaction(a “multiplex” assay). In view of the above, one skilled in the artrealizes that the methods disclosed herein may be practiced using one orany combination of the assays described above or any another geneticassay.

Labeling

In some embodiments, the protein, polypeptide, nucleic acid, fragmentsthereof, or fragments thereof ligated to adaptor regions used in theaspects disclosed herein are detectably labeled. As a non-limitingexample, a nucleic acid sequence hybridizable to a nucleic acid sequencecomprising the SNP within rs5745994 is detectably labeled. As anon-limiting example, a nucleic acid sequence hybridizable to a nucleicacid sequence comprising the SNP within rs11757159 is detectablylabeled. For example, the detectable label can be a fluorescent label,e.g., by incorporation of nucleotide analogues. Other labels suitablefor use in aspects disclosed herein include, but are not limited to,biotin, iminobiotin, antigens, cofactors, dinitrophenol, lipoic acid,olefinic compounds, detectable polypeptides, electron rich molecules,enzymes capable of generating a detectable signal by action upon asubstrate, and radioactive isotopes.

Radioactive isotopes include that can be used in conjunction with theaspects disclosed herein, but are not limited to, ³²P and ¹⁴C.Fluorescent molecules suitable for aspects disclosed herein include, butare not limited to, fluorescein and its derivatives, rhodamine and itsderivatives, texas red, 5′carboxy-fluorescein (“FAM”), 2′,7′-dimethoxy-4′, 5′-dichloro-6-carboxy-fluorescein (“JOE”), N, N, N′,N′-tetramethyl-6-carboxy-rhodamine (“TAMRA”), 6-carboxy-X-rhodamine(“ROX”), HEX, TET, IRD40, and IRD41.

Fluorescent molecules which are suitable for use according to theaspects disclosed herein further include: cyamine dyes, including butnot limited to Cy2, Cy3, Cy3.5, CY5, Cy5.5, Cy7 and FLUORX; BODIPY dyesincluding but not limited to BODIPY-FL, BODIPY-TR, BODIPY-TMR,BODIPY-630/650, and BODIPY-650/670; and ALEXA dyes, including but notlimited to ALEXA-488, ALEXA-532, ALEXA-546, ALEXA-568, and ALEXA-594; aswell as other fluorescent dyes which will be known to those who areskilled in the art. Electron rich indicator molecules suitable for theaspects disclosed herein include, but are not limited to, ferritin,hemocyanin and colloidal gold.

Two-color fluorescence labeling and detection schemes may also be used(Shena et al., 1995, Science 270:467-470). Use of two or more labels canbe useful in detecting variations due to minor differences inexperimental conditions (e.g., hybridization conditions). In someembodiments, at least 5, 10, 20, or 100 dyes of different colors can beused for labeling. Such labeling would also permit analysis of multiplesamples simultaneously which is encompassed by aspects disclosed herein.

The labeled nucleic acid samples, fragments thereof, or fragmentsthereof ligated to adaptor regions that can be used in the aspectsdisclosed herein are contacted to a plurality of oligonucleotide probesunder conditions that allow sample nucleic acids having sequencescomplementary to the probes to hybridize thereto. Depending on the typeof label used, the hybridization signals can be detected using methodswell known to those of skill in the art including, but not limited to,X-Ray film, phosphor imager, or CCD camera. When fluorescently labeledprobes are used, the fluorescence emissions at each site of a transcriptarray can be, preferably, detected by scanning confocal lasermicroscopy. In one embodiment, a separate scan, using the appropriateexcitation line, is carried out for each of the two fluorophores used.Alternatively, a laser can be used that allows simultaneous specimenillumination at wavelengths specific to the two fluorophores andemissions from the two fluorophores can be analyzed simultaneously (seeShalon et al. (1996) Genome Res. 6, 639-645). In a preferred embodiment,the arrays are scanned with a laser fluorescence scanner with a computercontrolled X-Y stage and a microscope objective. Sequential excitationof the two fluorophores is achieved with a multi-line, mixed gas laser,and the emitted light is split by wavelength and detected with twophotomultiplier tubes. Such fluorescence laser scanning devices aredescribed, e.g., in Schena et al. (1996) Genome Res. 6, 639-645.Alternatively, a fiber-optic bundle can be used such as that describedby Ferguson et al. (1996) Nat. Biotech. 14, 1681-1684. The resultingsignals can then be analyzed to determine the expression of TNFRSF1B,ANCA, TNFR2 and/or HLA-DRB6 and housekeeping genes, using computersoftware.

In other embodiments, where genomic DNA of a subject is fragmented usingrestriction endonucleases and amplified prior to analysis, theamplification can comprise cloning regions of genomic DNA of thesubject. In such methods, amplification of the DNA regions is achievedthrough the cloning process. For example, expression vectors can beengineered to express large quantities of particular fragments ofgenomic DNA of the subject (Sambrook and Russel, Molecular Cloning: ALaboratory Manual 4^(th) ed., Cold Spring Harbor Laboratory Press (ColdSpring Harbor, N.Y. 2012)).

In yet other embodiments, where the DNA of a subject is fragmented usingrestriction endonucleases and amplified prior to analysis, theamplification comprises expressing a nucleic acid encoding a gene, or agene and flanking genomic regions of nucleic acids, from the subject.RNA (pre-messenger RNA) that comprises the entire transcript includingintrons is then isolated and used in the methods disclosed herein toanalyze and provide a genetic signature of a cancer. In certainembodiments, no amplification is required. In such embodiments, thegenomic DNA, or pre-RNA, of a subject may be fragmented usingrestriction endonucleases or other methods. The resulting fragments maybe hybridized to SNP probes. Typically, greater quantities of DNA areneeded to be isolated in comparison to the quantity of DNA or pre-mRNAneeded where fragments are amplified. For example, where the nucleicacid of a subject is not amplified, a DNA sample of a subject for use inhybridization may be about 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900ng, or 1000 ng of DNA or greater. Alternatively, in other embodiments,methods are used that require very small amounts of nucleic acids foranalysis, such as less than 400 ng, 300 ng, 200 ng, 100 ng, 90 ng, 85ng, 80 ng, 75 ng, 70 ng, 65 ng, 60 ng, 55 ng, 50 ng, or less, such as isused for molecular inversion probe (MIP) assays. These techniques areparticularly useful for analyzing clinical samples, such as paraffinembedded formalin-fixed material or small core needle biopsies,characterized as being readily available but generally having reducedDNA quality (e.g., small, fragmented DNA) and/or not providing largeamounts of nucleic acids.

Once the expression levels have been determined, the resulting data canbe analyzed using various algorithms, based on well-known methods usedby those skilled in the art.

Systems

Disclosed herein, in some embodiments, is a system for detectinganti-TNF non-response in a subject, comprising analyzing genes or geneproducts expressed from TNFRSF1B, and/or HLA-DRB6, in a biologicalsample obtained from a subject. In some embodiments, the SNPs rs5745994and rs11757159 of TNFRSF1B, and/or HLA-DRB6, respectively, are analyzed.The system is configured to implement the methods described in thisdisclosure, including, but not limited to, analyzing genes or geneexpression products from the genes of a subject to determine whether thesubject is, or is susceptible to being, non-responsive to anti-TNFtherapy.

In some embodiments, disclosed herein is a system for detecting anti-TNFnon-response in a subject, comprising: (a) a computer processing device,optionally connected to a computer network; and (b) a software moduleexecuted by the computer processing device to analyze a gene or geneexpression product from one or more of the following TNFRSF1B, and/orHLA-DRB6, in a biological sample from a subject. In some instances, thesystem comprises a central processing unit (CPU), memory (e.g., randomaccess memory, flash memory), electronic storage unit, computer program,communication interface to communicate with one or more other systems,and any combination thereof. In some instances, the system is coupled toa computer network, for example, the Internet, intranet, and/or extranetthat is in communication with the Internet, a telecommunication, or datanetwork. In some embodiments, the system comprises a storage unit tostore data and information regarding any aspect of the methods describedin this disclosure. Various aspects of the system are a product orarticle or manufacture.

One feature of a computer program includes a sequence of instructions,executable in the digital processing device's CPU, written to perform aspecified task. In some embodiments, computer readable instructions areimplemented as program modules, such as functions, features, ApplicationProgramming Interfaces (APIs), data structures, and the like, thatperform particular tasks or implement particular abstract data types. Inlight of the disclosure provided herein, those of skill in the art willrecognize that a computer program may be written in various versions ofvarious languages.

The functionality of the computer readable instructions are combined ordistributed as desired in various environments. In some instances, acomputer program comprises one sequence of instructions or a pluralityof sequences of instructions. A computer program may be provided fromone location. A computer program may be provided from a plurality oflocations. In some embodiment, a computer program includes one or moresoftware modules. In some embodiments, a computer program includes, inpart or in whole, one or more web applications, one or more mobileapplications, one or more standalone applications, one or more webbrowser plug-ins, extensions, add-ins, or add-ons, or combinationsthereof.

Web Application

In some embodiments, a computer program includes a web application. Inlight of the disclosure provided herein, those of skill in the art willrecognize that a web application may utilize one or more softwareframeworks and one or more database systems. A web application, forexample, is created upon a software framework such as Microsoft® .NET orRuby on Rails (RoR). A web application, in some instances, utilizes oneor more database systems including, by way of non-limiting examples,relational, non-relational, feature oriented, associative, and XMLdatabase systems. Suitable relational database systems include, by wayof non-limiting examples, Microsoft® SQL Server, mySQL™, and Oracle®.Those of skill in the art will also recognize that a web application maybe written in one or more versions of one or more languages. In someembodiments, a web application is written in one or more markuplanguages, presentation definition languages, client-side scriptinglanguages, server-side coding languages, database query languages, orcombinations thereof. In some embodiments, a web application is writtento some extent in a markup language such as Hypertext Markup Language(HTML), Extensible Hypertext Markup Language (XHTML), or eXtensibleMarkup Language (XML). In some embodiments, a web application is writtento some extent in a presentation definition language such as CascadingStyle Sheets (CSS). In some embodiments, a web application is written tosome extent in a client-side scripting language such as AsynchronousJavascript and XML (AJAX), Flash® Actionscript, Javascript, orSilverlight®. In some embodiments, a web application is written to someextent in a server-side coding language such as Active Server Pages(ASP), ColdFusion®, Perl, Java™, JavaServer Pages (JSP), HypertextPreprocessor (PHP), Python™, Ruby, Tcl, Smalltalk, WebDNA®, or Groovy.In some embodiments, a web application is written to some extent in adatabase query language such as Structured Query Language (SQL). A webapplication may integrate enterprise server products such as IBM® LotusDomino®. A web application may include a media player element. A mediaplayer element may utilize one or more of many suitable multimediatechnologies including, by way of non-limiting examples, Adobe® Flash®,HTML 5, Apple® QuickTime®, Microsoft® Silverlight®, Java™, and Unity®.

Mobile Application

In some instances, a computer program includes a mobile applicationprovided to a mobile digital processing device. The mobile applicationmay be provided to a mobile digital processing device at the time it ismanufactured. The mobile application may be provided to a mobile digitalprocessing device via the computer network described herein.

A mobile application is created by techniques known to those of skill inthe art using hardware, languages, and development environments known tothe art. Those of skill in the art will recognize that mobileapplications may be written in several languages. Suitable programminglanguages include, by way of non-limiting examples, C, C++, C #,Featureive-C, Java™, Javascript, Pascal, Feature Pascal, Python™, Ruby,VB.NET, WML, and XHTML/HTML with or without CSS, or combinationsthereof.

Suitable mobile application development environments are available fromseveral sources. Commercially available development environmentsinclude, by way of non-limiting examples, AirplaySDK, alcheMo,Appcelerator®, Celsius, Bedrock, Flash Lite, .NET Compact Framework,Rhomobile, and WorkLight Mobile Platform. Other development environmentsmay be available without cost including, by way of non-limitingexamples, Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile devicemanufacturers distribute software developer kits including, by way ofnon-limiting examples, iPhone and iPad (iOS) SDK, Android™ SDK,BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, andWindows® Mobile SDK.

Those of skill in the art will recognize that several commercial forumsare available for distribution of mobile applications including, by wayof non-limiting examples, Apple® App Store, Android™ Market, BlackBerry®App World, App Store for Palm devices, App Catalog for webOS, Windows®Marketplace for Mobile, Ovi Store for Nokia® devices, Samsung® Apps, andNintendo® DSi Shop.

Standalone Application

In some embodiments, a computer program includes a standaloneapplication, which is a program that may be run as an independentcomputer process, not an add-on to an existing process, e.g., not aplug-in. Those of skill in the art will recognize that standaloneapplications are sometimes compiled. In some instances, a compiler is acomputer program(s) that transforms source code written in a programminglanguage into binary feature code such as assembly language or machinecode. Suitable compiled programming languages include, by way ofnon-limiting examples, C, C++, Featureive-C, COBOL, Delphi, Eiffel,Java™, Lisp, Python™, Visual Basic, and VB .NET, or combinationsthereof. Compilation may be often performed, at least in part, to createan executable program. In some instances, a computer program includesone or more executable complied applications.

Web Browser Plug-in

A computer program, in some aspects, includes a web browser plug-in. Incomputing, a plug-in, in some instances, is one or more softwarecomponents that add specific functionality to a larger softwareapplication. Makers of software applications may support plug-ins toenable third-party developers to create abilities which extend anapplication, to support easily adding new features, and to reduce thesize of an application. When supported, plug-ins enable customizing thefunctionality of a software application. For example, plug-ins arecommonly used in web browsers to play video, generate interactivity,scan for viruses, and display particular file types. Those of skill inthe art will be familiar with several web browser plug-ins including,Adobe® Flash® Player, Microsoft® Silverlight®, and Apple® QuickTime®.The toolbar may comprise one or more web browser extensions, add-ins, oradd-ons. The toolbar may comprise one or more explorer bars, tool bands,or desk bands.

In view of the disclosure provided herein, those of skill in the artwill recognize that several plug-in frameworks are available that enabledevelopment of plug-ins in various programming languages, including, byway of non-limiting examples, C++, Delphi, Java™, PHP, Python™, and VB.NET, or combinations thereof.

In some embodiments, Web browsers (also called Internet browsers) aresoftware applications, designed for use with network-connected digitalprocessing devices, for retrieving, presenting, and traversinginformation resources on the World Wide Web. Suitable web browsersinclude, by way of non-limiting examples, Microsoft® Internet Explorer®,Mozilla® Firefox®, Google® Chrome, Apple® Safari®, Opera Software®Opera®, and KDE Konqueror. The web browser, in some instances, is amobile web browser. Mobile web browsers (also called mircrobrowsers,mini-browsers, and wireless browsers) may be designed for use on mobiledigital processing devices including, by way of non-limiting examples,handheld computers, tablet computers, netbook computers, subnotebookcomputers, smartphones, music players, personal digital assistants(PDAs), and handheld video game systems. Suitable mobile web browsersinclude, by way of non-limiting examples, Google® Android® browser, RIMBlackBerry® Browser, Apple® Safari®, Palm® Blazer, Palm® WebOS® Browser,Mozilla® Firefox® for mobile, Microsoft® Internet Explorer® Mobile,Amazon® Kindle® Basic Web, Nokia® Browser, Opera Software® Opera®Mobile, and Sony® PSP™ browser.

Software Modules

The medium, method, and system disclosed herein comprise one or moresoftwares, servers, and database modules, or use of the same. In view ofthe disclosure provided herein, software modules may be created bytechniques known to those of skill in the art using machines, software,and languages known to the art. The software modules disclosed hereinmay be implemented in a multitude of ways. In some embodiments, asoftware module comprises a file, a section of code, a programmingfeature, a programming structure, or combinations thereof. A softwaremodule may comprise a plurality of files, a plurality of sections ofcode, a plurality of programming features, a plurality of programmingstructures, or combinations thereof. By way of non-limiting examples,the one or more software modules comprises a web application, a mobileapplication, and/or a standalone application. Software modules may be inone computer program or application. Software modules may be in morethan one computer program or application. Software modules may be hostedon one machine. Software modules may be hosted on more than one machine.Software modules may be hosted on cloud computing platforms. Softwaremodules may be hosted on one or more machines in one location. Softwaremodules may be hosted on one or more machines in more than one location.

Databases

The medium, method, and system disclosed herein comprise one or moredatabases, or use of the same. In view of the disclosure providedherein, those of skill in the art will recognize that many databases aresuitable for storage and retrieval of geologic profile, operatoractivities, division of interest, and/or contact information of royaltyowners. Suitable databases include, by way of non-limiting examples,relational databases, non-relational databases, feature orienteddatabases, feature databases, entity-relationship model databases,associative databases, and XML databases. In some embodiments, adatabase is internet-based. In some embodiments, a database isweb-based. In some embodiments, a database is cloud computing-based. Adatabase may be based on one or more local computer storage devices.

Data Transmission

The subject matter described herein, including methods for obtaining andanalyzing a molecular signature from a subject having a pigmented skinlesion, methods for obtaining a pigmented skin lesion, correspondingtransmission of data, in certain aspects, are configured to be performedin one or more facilities at one or more locations. Facility locationsare not limited by country and include any country or territory. In someinstances, one or more steps for obtaining a molecular signature from asample are performed in a different country than another step of themethod. In some instances, one or more steps for obtaining a sample areperformed in a different country than one or more steps for obtaining amolecular signature from a sample. In some embodiments, one or moremethod steps involving a computer system are performed in a differentcountry than another step of the methods provided herein. In someembodiments, data processing and analyses are performed in a differentcountry or location than one or more steps of the methods describedherein. In some embodiments, one or more articles, products, or data aretransferred from one or more of the facilities to one or more differentfacilities for analysis or further analysis. An article includes, but isnot limited to, one or more components obtained from the tape strippingmethods such as the adhesive tape, isolated cellular material obtainedfrom an adhesive tape, processed cellular material, data, and anyarticle or product disclosed herein as an article or product. Processedcellular material includes, but is not limited to, cDNA reversetranscribed from RNA, amplified RNA, amplified cDNA, sequenced DNA,isolated and/or purified RNA, isolated and/or purified DNA, and isolatedand/or purified polypeptide. Data includes, but is not limited to,information regarding the gene expression profile of one or more targetgenes, information regarding a gene sequence profile signature,information regarding a protein sequence profile, information regardingthe characteristic of a pigmented skin lesion (e.g., non-melanoma,melanoma in situ, invasive melanoma, stage 1 melanoma, stage 2 melanoma,stage 3 melanoma, stage 4 melanoma), and any data produced by themethods disclosed herein. In some embodiments of the methods and systemsdescribed herein, the analysis is performed and a subsequent datatransmission step will convey or transmit the results of the analysis.Information regarding a pigmented skin lesion includes, but is notlimited to, identification of melanoma, likelihood of treatment successfor a subject having melanoma, identification of progression of amelanoma, identification of melanoma in situ, identification of invasivemelanoma, and identification of a melanoma stage (e.g., 0, 1, 2, 3, 4).

In some embodiments, any step of any method described herein isperformed by a software program or module on a computer. In additionalor further embodiments, data from any step of any method describedherein is transferred to and from facilities located within the same ordifferent countries, including analysis performed in one facility in aparticular location and the data shipped to another location or directlyto an individual in the same or a different country. In additional orfurther embodiments, data from any step of any method described herein(including characterization of melanoma in situ and/or invasivemelanoma, information regarding cellular material such as DNA, RNA, andprotein as well as transformed data, e.g. a molecular signature, fromcellular material) is transferred to and/or received from a facilitylocated within the same or different countries, including analysis of adata input, such as cellular material, performed in one facility in aparticular location and corresponding data transmitted to anotherlocation, or directly to an individual, such as data related to thediagnosis, prognosis, responsiveness to therapy, or the like, in thesame or different location or country.

EXAMPLES

The following examples are not intended to limit the scope of the claimsto the aspects disclosed herein, but are rather intended to be exemplaryof certain embodiments. Any variations in the exemplified methods whichoccur to the skilled artisan are intended to fall within the scope ofthe aspects disclosed herein.

Example 1

Cedars-Sinai Cohort

Subjects were recruited in accordance with the recruitment carried outin Franke et al., Genome-wide meta-analysis increases to 71 the numberof confirmed Crohn's disease susceptibility loci. Nat Genet. 2010;42:1118-1125; McGovern et al., Fucosyltransferase 2 (FUT2) non-secretorstatus is associated with Crohn's disease. Hum Mol Genet. 2010;19:3468-3476; Haritunians T et al., Genetic predictors of medicallyrefractive ulcerative colitis, Inflamm Bow Dis. 2010; 16:1830-1840 andAnderson et al., Meta-analysis identifies 29 additional ulcerativecolitis risk loci, increasing the number of confirmed associations to47. Nat Genet. 2011; 43:246-252.

3110 patients with IBD were recruited at the Cedars-Sinai InflammatoryBowel Disease Centers from 1985 to 2010. The diagnosis of each patientwas based on standard endoscopic, histologic, and radiographic featureas previously described (Mow et al., Association of antibody responsesto microbial antigens and complications of small bowel Crohn's disease.Gastroenterology. 2004; 126:414-424). Blood samples were collected atthe time of enrollment. All study participants gave written informedcontent and the study protocol and data collection, as well as DNApreparation/genotyping and antibody measurement, was approved by theCedars-Sinai Medical Center's Institutional Review Board.

Mount Sinai Hospital Cohort

A total of 1853 IBD cases were recruited and similarly characterized atMount Sinai Hospital in Toronto, Canada. All subjects provided writtenconsent after institutional review board's approval. The diagnosis ofeach patient was based on standard endoscopic, histologic, andradiographic feature.

ANCA Level Measurement

ANCA levels in serum from subjects from both centers were measured byenzyme-linked immunosorbent assay as previously described (Mow et al.,Association of antibody responses to microbial antigens andcomplications of small bowel Crohn's disease. Gastroenterology. 2004;126:414-424). All sera were analyzed in a blinded fashion atCedars-Sinai Medical Center. Antibody levels were determined and resultsexpressed as enzyme-linked immunosorbent assay units (EU/mL) as comparedwith a positive control (Mow et al., Association of antibody responsesto microbial antigens and complications of small bowel Crohn's disease.Gastroenterology. 2004; 126:414-424). Qualitative positivity to ANCA wasdefined as being greater than cutoff values greater than 2 SDs abovemean control titers.

Whole-Genome Genotyping:

Cedars-Sinai Cohort

Genotyping was performed at Cedars-Sinai Medical Center using Illuminawhole-genome arrays per manufacturer's protocol (Illumina, San Diego,Calif.). The discovery cohort was genotyped on 3 platforms, includingIllumina HumanCNV370-Quad (830 subjects), Human610-Quad (1037 subjects),and HumanOmniExpress (1243 subjects) arrays (total 3110 independentsubjects). Average genotyping call rates for samples that passed qualitycontrol (QC) were 99.86% (HumanCNV370-Quad), 99.83% (Human610-Quad), and99.85% (HumanOmniExpress). One to two percent of samples were genotypedin replicate and yielded average 99.99% concordance for genotypescalled. Optimal allele-calling was verified by manual review of topassociated single-nucleotide polymorphisms (SNPs).

A stringent QC procedure was applied to the GWAS data. Of the 3110subjects genotyped, 10 were removed because of high missing rate (>2%),27 were removed because of cryptic relatedness (Pi_Hat>0.05), 3 wereremoved because sample either withdrew from the study or was laterreclassified as non-IBD, leaving 3070 individuals for further analysis.Seventy-six individuals identified as nonwhites by principal componentanalysis were also removed. A total of 2959 of the 2994 genotypedsubjects had ANCA status and were thereby included in the analyses.

Mount Sinai Hospital Cohort

A total of 1853 patients with IBD from the Mount Sinai Hospital Cohortin Toronto were genotyped on Illumina HumanOmniExpress array. Averagegenotyping call rates for samples that passed QC were 99.88%. Thirty-twosamples genotyped in replicate and yielded average 99.99% concordancefor genotypes called. After similar QC procedures as the discoverycohort, 1834 subjects remain in the cleaned data set, of which 419 hadANCA status available measured in the same laboratory as the discoverycohort. The 419 individuals with consistent ANCA measurements wereincluded in this study as replication cohort.

Imputation

To consolidate data from different genotyping platforms, imputation wasperformed using a hidden Markov model-based algorithm available in theIMPUTE224 software package with Phase 2 HapMap genotypes as the Ref. 25.In the imputed data set, only imputed SNPs with minor allele frequency(MAF)>0.01 and quality score >0.80 in all 3 Illumina platforms wereretained, leaving approximately 2.01M SNPs for analyses.

Validation of Top-Hit SNP rs5745994

The most significant novel association observed in the genome-wideassociation analysis was with an imputed SNP (rs5745994). To validatethe imputation, this SNP was included as custom content on the IlluminaHumanExome+ array. BeadChip and genotyped as part of a larger project atCedars-Sinai Medical Center following manufacturer's protocol (Illumina,San Diego, Calif.). Genotype clusters for rs5745994 were visualized toensure accurate allele-calling. The average genotyping rate of samplesin the project that passed genotyping QC was 99.98%. Two hundredseventy-three samples performed in replicate as controls yielded99.9963% concordance for genotypes called.

Measure of TNFR1 and TNFR2 Levels

TNFR1 and TNFR2 concentrations were assessed in serum by commerciallyavailable enzyme-linked immunosorbent assay kits according to themanufacturer specified protocol (Biosource Invitrogen, KAC1761,KAC1771). The assay laboratory at Cedars-Sinai was blinded to subjectstatus. Concentrations are reported in nanogram per milliliter.

Statistical Analysis

ANCA level in IBD cases is not normally distributed and the traditionalmethod for analyzing skewed data, the nonparametric Mann-Whitney U test,is known to have lower efficiency and cannot incorporate covariates inanalyses. Therefore, linear regression was used with the nontransformedANCA level as outcome and for top SNPs, a permutation test was used toretain the type I error rate. The top 3 principal components andgenotyping platform (in the discovery cohort) were included in theanalysis as covariates to control for potential confounding. SNPTEST2.2027 and R3.0228 were used in the statistical analysis. Similarly,permutation test was also performed in the analysis comparingcirculating levels of TNFR1 and TNFR2 in carriers and non-carriers ofthe TNFRSF1B SNP.

Demographic Characteristics

Table 4 shows the demographic characteristics of the 2959 individualsincluded in the final analyses of the discovery cohort of 1653 (55.86%)CD, 1193 (40.31%) UC, and 113 (3.82%) IBD-unclassified. The average ageof disease onset was 25.65±14.56 years old and 1513 (51.13%) are men.Median ANCA levels were 13.21 in CD, 36.00 in UC, and 18.93 in all IBD.A total of 433 CD (26.19%), 788 UC (66.05%), and 1278 all IBD (43.19%)were ANCA positive.

TABLE 4 Demographic Characters of the Discovery Cohort CD UC All IBD N1653 1193 2959^(a)   Sex (M/F) 842/811 608/585 1513/1446 Age ofdiagnosis 23.64 ± 13.73 28.19 ± 15.05 25.65 ± 14.56 ANCA level (median)13.21 36.00 18.93 ANCA +/− 433/1220 788/405 1278/1681 ^(a)One hundredthirteen IBD undetermined patients were also included.SNP Associations with ANCA Level

Overall genomic inflation factor for the genome-wide association in thediscovery cohort was 1.023 (see FIG. 1 and FIG. 2). FIG. 1 shows theManhattan plot for association with ANCA level in the GWAS. Beforeperforming permutation testing, 2 signals were observed that reachedstandard and stringent criteria for genome-wide significance thresholdof 5.0×10⁻⁸. The most significant SNP (rs11757159, MAF=0.326) is locatedin HLA-DRB6, with the minor allele C (frequency 0.33) associated withlower ANCA level (beta=−7.09, 95% CI, −9.27 to −4.91), with apermutation test P value of 2.55×10⁻¹⁰. The second signal achievinggenome-wide significance was located on 1p36.22 (rs5745994, MAF=0.028)which is located in the intron of TNFRSF1B. Individuals carrying theminor allele (C allele) have higher ANCA level (beta=18.12, 95% CI,11.82-24.22), with a P-value of 1.89×10⁻⁸ after permutation testing(Table 5). Additional haplotype-based analyses and conditional analysesfor this region suggest that there is only 1 signal at this locus,tagged by rs5745994 (data not shown).

TABLE 5 Top SNPs in GWAS Analysis Discovery Cohort Replication Cohort (N= 2959) (N = 419) Meta-Analysis Beta Beta Beta SEQ. SNP CHR PositionGene A1 A2 MAF (95% CI) P^(a) MAF (95% CI) P^(a) (95% CI) P^(a) 1rs11757159 6 32.628250 HLA-DRB6 T C 0.326 −7.09 2.55 × 0.294 −2.30 0.262−5.99 3.25 × (−9.27 10⁻¹⁰ (−6.28 (−7.91 10⁻⁸ to −4.91) to 1.69) to−4.07) 2 rs5745994 1 12.169715 TNFR SF1B T C 0.028 18.12 1.89 × 0.03116.91 2.38 × 17.81 8.97 × (11.86 10⁻⁸ (6.13 10⁻³ (12.36 10⁻¹⁰ to 24.38)to 27.69) to 23.25) ^(a)P values after permutation test. CHR,chromosome; MAF, minor allele frequency.TNFSF1B SNP rs5745994 Associated with IBD

The association of the novel signal in TNFRSF1B was further examinedseparately in CD and UC. The signal is observed in both CD and UC, witha stronger effect observed in UC (CD: beta=11.14, 95% CI, 4.67-17.62,P=1.24×10³; UC: beta=23.83, 95% CI, 12.88-34.78, P=8.42×10⁻⁵).

Validation of the TNFRSF1B SNP in ExomeChip

As the TNFRSF1B SNP (rs5745994) was an imputed SNP, the imputation, byincorporating this SNP as a SNP in the customized content in ourExomeChip genotyping efforts were validated. Concordance rate betweenimputed and genotyped data for 2841 individuals with both GWAS andExomeChip data was 99.64%. As expected, association analysis using onlygenotyped SNPs yielded similar results (beta=16.78, 95% CI, 10.16-23.40,P=7.15×10⁻⁷).

Replication of the Top Signals

Using an additional cohort of 419 patients with IBD from Mount SinaiHospital in Toronto in which ANCA levels were analyzed at Cedars-Sinai,the association of the TNFRSF1B with ANCA levels were further examined(Table 2). The original observation of an association between the Callele of rs5745994 and ANCA level, with similar effect magnitude(beta=16.91, 95% CI, 6.13-27.69, P=2.38×10⁻³), was confirmed. With aQ-value of 0.036, a fixed-effect meta-analysis for the association ofrs5745994 in both cohorts was performed and observed a strongerassociation signal (beta=17.81, 95% CI, 12.36-23.25, P=8.97×10¹⁰). TheHLA signal observed in the discovery cohort failed to replicate(beta=−2.30, P=0.262).

Association of the TNFRSF1B SNP with TNFR2 Level

Next, the association between TNFRSF1B (rs5745994) and serum TNFR1 (ascontrol) and TNFR2 levels was tested, in a subset of 239 patients withIBD, of which 58 carried the C allele of rs5745994 with the remainderbeing homozygous for the T allele. FIG. 3 shows the association ofrs5745994 with TNFR1 and TNFR2. TNFRSF1B was not associated with TNFR1(median 2.99 EU/mL in C allele carriers compared with 3.06 EU/mL innoncarriers, P=0.71). In contrast, the median TNFR2 was 8.23 EU/mL incarriers and 9.12 EU/mL in noncarriers (P=0.033), indicating lower serumTNFR2 level in carriers of the C allele.

Example 2

Detecting Anti-TNF-Alpha Non-Response as Determined by ANCA Level

Subjects are selected based on a diagnosis of IBD. The diagnosis of eachpatient is based on standard endoscopic, histologic, and radiographicfeature as previously described (Mow et al., Association of antibodyresponses to microbial antigens and complications of small bowel Crohn'sdisease. Gastroenterology. 2004; 126:414-424). Blood samples arecollected. Blood sera or plasma are isolated from blood samples.

Fixed-granulocyte ELISA assay: ANCA levels in blood sera or plasma aremeasured using a high-binding polystyrene microtiter plates coated witha monolayer of granulocytes by the addition of 0.1 ml of Hanks' balancedbuffered-sale solution containing 500,000 granulocytes. After the cellssettle and spread for 30 minutes at 23° C., the plates are centrifugedat 1000 rpm (300 g) for 5 minutes, the supernatant is aspirated from thewells, and the plates are air-dried for 2 hours. Methanol (100%) is thenadded for 10 minutes and discarded. The plates are air-dried and thenstored at −20° C. For use, the plates are brought to room temperatureand blocked for nonspecific binding by addition of 150 microliters of 1%BSA and 1% PBS for 1 hour. The blocking material is discarded, and then100 microliters of solution of test serum diluted in 1% BSA/PBS is addedand incubated for 1 hour in a humidified box. After four washes with PBSand Tween, 100 microliter per well of a 1:1500 dilution of alkalinephosphatase-coupled goat antibuman gamma-chain specific antibody (Tago,Inc., Burlingame, Calif.) in 1% BSA/PBS is added per well for 1 hour.This antibody is discarded, and the wells are washed three times withPBS/Tween and four times with Tris/NaCl (0.05 mol/L of Tris base in 0.9N NaCl, pH 7.5). Substrate solution (disodium P-nitrophenol phosphate,1.5 mg/ml and 100 p.1 per well) is added, and color development isallowed to proceed until absorbance at 405 nm in the positive controlwells was 0.8. Levels are determined relative to a standard comprisingof pooled sera obtained from well-characterized pANCA ulcerative colitispatients. Results are expressed as ELISA units, which reflect apercentage of the standard. Sera with circulating antineutrophilcytoplasmic IgG antibody exceeding the about 100 EU are termed ANCA+.Optionally, the subject is treated with a non-anti-TNF therapeutic agentprovided herein. In some embodiments, the therapeutic agent comprises ananti-TL1A antibody.

Indirect immunofluorescence assay: Neutrophils separated by dextransedimentation are re-suspended in PBS, and 100,000 cells are prepared onslides by cytocentrifugation (Cytospin, Shandon Southern Products,Cheshire, U.K.). The slides are fixed in 100% methanol at 4° C. for 10minutes, air-dried, and stored at −20° C. The sera are tested at adilution of 1:20, and staining with fluoroscein-labeled F(ab′)₂gamma-chain-specific antibody is performed as described. Slides wereexamined by fluorescence microscopy (40×Epifluor lens, Carl Zeiss, Inc.,Thornwood, N.Y.).

Example 3

Detecting Anti-TNF-Alpha Non-Response as Determined by Presence ofTNFRSF1B SNP

Subjects are selected based on a diagnosis of IBD. The diagnosis of eachpatient is based on standard endoscopic, histologic, and radiographicfeature as previously described (Mow et al., Association of antibodyresponses to microbial antigens and complications of small bowel Crohn'sdisease. Gastroenterology. 2004; 126:414-424). Blood samples arecollected.

The presence of the TNFRSF1B SNP rs5745994 is detected usingquantitative nucleic acid amplification (qPCR) protocols, such as Innis,et al. (1990) PCR Protocols, A Guide to Methods and Applications,Academic Press, Inc. N.Y.). The measurement of DNA copy number atmicrosatellite loci using qPCR analysis is performed as described inGinzonger, et al. (2000) Cancer Research 60:5405-5409. The probesequence used is sufficiently complimentary to the oligonucleotidesequence of the TNFRSF1B SNP rs5745994 to allow detection of the genecomprising the risk allele, “C,” at position 256 within rs5745994. Theprobe sequence is conjugated to a reporter dye, such as TaqMan or SYBRgreen, and a quencher, such that hybridization of the dye-conjugatedprobe sequence to the TNFRSF1B SNP rs5745994 is visualized.

Example 4

Detecting Anti-TNF-Alpha Non-Response as Determined by TNFR2 Level

Subjects are selected based on a diagnosis of IBD. The diagnosis of eachpatient is based on standard endoscopic, histologic, and radiographicfeature as previously described (Mow et al., Association of antibodyresponses to microbial antigens and complications of small bowel Crohn'sdisease. Gastroenterology. 2004; 126:414-424). Blood samples arecollected. Blood sera or plasma are isolated from the blood samples.

TNFR2 level is assessed in serum or plasma by commercially availableenzyme-linked immunosorbent assay kits according to the manufacturerspecified protocol (Biosource Invitrogen, KAC1761, KAC1771).Concentrations are reported in nanogram per milliliter.

Example 5

Detecting Anti-TNF-Alpha Non-Response as Determined by Presence ofTNFRSF1B SNP and ANCA Level

Subjects are selected based on a diagnosis of IBD. The diagnosis of eachpatient is based on standard endoscopic, histologic, and radiographicfeature as previously described (Mow et al., Association of antibodyresponses to microbial antigens and complications of small bowel Crohn'sdisease. Gastroenterology. 2004; 126:414-424). Blood samples arecollected. Blood sera or plasma are isolated from the blood samples.

The presence of the TNFRSF1B SNP rs5745994 is detected usingquantitative nucleic acid amplification (qPCR) protocols, such as Innis,et al. (1990) PCR Protocols, A Guide to Methods and Applications,Academic Press, Inc. N.Y.). The measurement of DNA copy number atmicrosatellite loci using qPCR analysis is performed as described inGinzonger, et al. (2000) Cancer Research 60:5405-5409. The probesequence used is sufficiently complimentary to the oligonucleotidesequence of the TNFRSF1B SNP rs5745994 to allow detection of the genecomprising the risk allele, “C,” at position 256 within rs5745994. Theprobe sequence is conjugated to a reporter dye, such as TaqMan or SYBRgreen, and a quencher, such that hybridization of the dye-conjugatedprobe sequence to the TNFRSF1B SNP rs5745994 is visualized.

Fixed-granulocyte ELSA assay: ANCA levels in blood sera or plasma aremeasured using a high-binding polystyrene microtiter plates coated witha monolayer of granulocyres by the addition of 0.1 ml of Hanks' balancedbuffered-sale solution containing 500,000 granulocytes. After the cellssettle and spread for 30 minutes at 23° C., the plates are centrifugedat 1000 rpm (300 g) for 5 minutes, the supernatant is aspirated from thewells, and the plates are air-dried for 2 hours. Methanol (100%) is thenadded for 10 minutes and discarded. The plates are air-dried and thenstored at −20° C. For use, the plates are brought to room temperatureand blocked for nonspecific binding by addition of 150 microliters of 1%BSA and 1% PBS for 1 hour. The blocking material is discarded, and then100 microliters of solution of test serum diluted in 1% BSA/PBS is addedand incubated for 1 hour in a humidified box. After four washes with PBSand Tween, 100 microliter per well of a 1:1500 dilution of alkalinephosphatase-coupled goat antibuman gamma-chain specific antibody (Tago,Inc., Burlingame, Calif.) in 1% BSA/PBS is added per well for 1 hour.This antibody is discarded, and the wells are washed three times withPBS/Tween and four times with Tris/NaCl (0.05 mol/L of Tris base in 0.9N NaCl, pH 7.5). Substrate solution (disodium P-nitrophenol phosphate,1.5 mg/ml and 100 p.1 per well) is added, and color development isallowed to proceed until absorbance at 405 nm in the positive controlwells was 0.8. Levels are determined relative to a standard comprisingof pooled sera obtained from well-characterized pANCA ulcerative colitispatients. Results are expressed as ELISA units, which reflect apercentage of the standard. Sera with circulating antineutrophilcytoplasmic IgG antibody that is at, or above about 50 EU are termedANCA+. Optionally, the subject is treated with a non-anti-TNFtherapeutic agent provided herein. In some embodiments, the therapeuticagent comprises an anti-TL1A antibody.

Indirect immunofluorescence assay: Neutrophils separated by dextransedimentation are re-suspended in PBS, and 100,000 cells are prepared onslides by cytocentrifugation (Cytospin, Shandon Southern Products,Cheshire, U.K.). The slides are fixed in 100% methanol at 4° C. for 10minutes, air-dried, and stored at −20° C. The sera are tested at adilution of 1:20, and staining with fluoroscein-labeled F(ab′)₂gamma-chain-specific antibody is performed as described. Slides wereexamined by fluorescence microscopy (40×Epifluor lens, Carl Zeiss, Inc.,Thornwood, N.Y.).

Example 6

Detecting Anti-TNF-Alpha Non-Response as Determined by Presence ofTNFRSF1B SNP and TNFR2 Level

Subjects are selected based on a diagnosis of IBD. The diagnosis of eachpatient is based on standard endoscopic, histologic, and radiographicfeature as previously described (Mow et al., Association of antibodyresponses to microbial antigens and complications of small bowel Crohn'sdisease. Gastroenterology. 2004; 126:414-424). Blood samples arecollected. Blood sera or plasma are isolated from the blood samples.

The presence of the TNFRSF1B SNP rs5745994 is detected usingquantitative nucleic acid amplification (qPCR) protocols, such as Innis,et al. (1990) PCR Protocols, A Guide to Methods and Applications,Academic Press, Inc. N.Y.). The measurement of DNA copy number atmicrosatellite loci using qPCR analysis is performed as described inGinzonger, et al. (2000) Cancer Research 60:5405-5409. The probesequence used is sufficiently complimentary to the oligonucleotidesequence of the TNFRSF1B SNP rs5745994 to allow detection of the genecomprising the risk allele, “C,” at position 256 within rs5745994. Theprobe sequence is conjugated to a reporter dye, such as TaqMan or SYBRgreen, and a quencher, such that hybridization of the dye-conjugatedprobe sequence to the TNFRSF1B SNP rs5745994 is visualized.

TNFR2 level is assessed in serum or plasma by commercially availableenzyme-linked immunosorbent assay kits according to the manufacturerspecified protocol (Biosource Invitrogen, KAC1761, KAC1771).Concentrations are reported in nanogram per milliliter.

Example 7

Detecting Anti-TNF-Alpha Non-Response as Determined by TNFR2 Level andANCA Level

Subjects are selected based on a diagnosis of IBD. The diagnosis of eachpatient is based on standard endoscopic, histologic, and radiographicfeature as previously described (Mow et al., Association of antibodyresponses to microbial antigens and complications of small bowel Crohn'sdisease. Gastroenterology. 2004; 126:414-424). Blood samples arecollected. Blood sera or plasma are isolated from the blood samples.

TNFR2 level is assessed in serum or plasma by commercially availableenzyme-linked immunosorbent assay kits according to the manufacturerspecified protocol (Biosource Invitrogen, KAC1761, KAC1771).Concentrations are reported in nanogram per milliliter.

Fixed-granulocyte ELISA assay: ANCA levels in blood sera or plasma aremeasured using a high-binding polystyrene microtiter plates coated witha monolayer of granulocyres by the addition of 0.1 ml of Hanks' balancedbuffered-sale solution containing 500,000 granulocytes. After the cellssettle and spread for 30 minutes at 23° C., the plates are centrifugedat 1000 rpm (300 g) for 5 minutes, the supernatant is aspirated from thewells, and the plates are air-dried for 2 hours. Methanol (100%) is thenadded for 10 minutes and discarded. The plates are air-dried and thenstored at −20° C. For use, the plates are brought to room temperatureand blocked for nonspecific binding by addition of 150 microliters of 1%BSA and 1% PBS for 1 hour. The blocking material is discarded, and then100 microliters of solution of test serum diluted in 1% BSA/PBS is addedand incubated for 1 hour in a humidified box. After four washes with PBSand Tween, 100 microliter per well of a 1:1500 dilution of alkalinephosphatase-coupled goat antibuman gamma-chain specific antibody (Tago,Inc., Burlingame, Calif.) in 1% BSA/PBS is added per well for 1 hour.This antibody is discarded, and the wells are washed three times withPBS/Tween and four times with Tris/NaCl (0.05 mol/L of Tris base in 0.9N NaCl, pH 7.5). Substrate solution (disodium P-nitrophenol phosphate,1.5 mg/ml and 100 p.1 per well) is added, and color development isallowed to proceed until absorbance at 405 nm in the positive controlwells was 0.8. Levels are determined relative to a standard comprisingof pooled sera obtained from well-characterized pANCA ulcerative colitispatients. Results are expressed as ELISA units, which reflect apercentage of the standard. Sera with circulating antineutrophilcytoplasmic IgG antibody that are at, or above, about 50 EU are termedANCA+. Optionally, the subject is treated with a non-anti-TNFtherapeutic agent provided herein. In some embodiments, the therapeuticagent comprises an anti-TL1A antibody.

Indirect immunofluorescence assay: Neutrophils separated by dextransedimentation are re-suspended in PBS, and 100,000 cells are prepared onslides by cytocentrifugation (Cytospin, Shandon Southern Products,Cheshire, U.K.). The slides are fixed in 100% methanol at 4° C. for 10minutes, air-dried, and stored at −20° C. The sera are tested at adilution of 1:20, and staining with fluoroscein-labeled F(ab′)₂gamma-chain-specific antibody is performed as described. Slides wereexamined by fluorescence microscopy (40×Epifluor lens, Carl Zeiss, Inc.,Thornwood, N.Y.).

Example 8

Detecting Anti-TNF-Alpha Non-Response as Determined by Presence ofTNFRSF1B SNP, ANCA Level, and TNFR2 Level

Subjects are selected based on a diagnosis of IBD. The diagnosis of eachpatient is based on standard endoscopic, histologic, and radiographicfeature as previously described (Mow et al., Association of antibodyresponses to microbial antigens and complications of small bowel Crohn'sdisease. Gastroenterology. 2004; 126:414-424). Blood samples arecollected. Blood sera or plasma are isolated from the blood samples.

The presence of the TNFRSF1B SNP rs5745994 is detected usingquantitative nucleic acid amplification (qPCR) protocols, such as Innis,et al. (1990) PCR Protocols, A Guide to Methods and Applications,Academic Press, Inc. N.Y.). The measurement of DNA copy number atmicrosatellite loci using qPCR analysis is performed as described inGinzonger, et al. (2000) Cancer Research 60:5405-5409. The probesequence used is sufficiently complimentary to the oligonucleotidesequence of the TNFRSF1B SNP rs5745994 to allow detection of the genecomprising the risk allele, “C,” at position 256 within rs5745994. Theprobe sequence is conjugated to a reporter dye, such as TaqMan or SYBRgreen, and a quencher, such that hybridization of the dye-conjugatedprobe sequence to the TNFRSF1B SNP rs5745994 is visualized.

TNFR2 level is assessed in serum or plasma by commercially availableenzyme-linked immunosorbent assay kits according to the manufacturerspecified protocol (Biosource Invitrogen, KAC1761, KAC1771).Concentrations are reported in nanogram per milliliter.

Fixed-granulocyte ELISA assay: ANCA levels in blood sera or plasma aremeasured using a high-binding polystyrene microtiter plates coated witha monolayer of granulocyres by the addition of 0.1 ml of Hanks' balancedbuffered-sale solution containing 500,000 granulocytes. After the cellssettle and spread for 30 minutes at 23° C., the plates are centrifugedat 1000 rpm (300 g) for 5 minutes, the supernatant is aspirated from thewells, and the plates are air-dried for 2 hours. Methanol (100%) is thenadded for 10 minutes and discarded. The plates are air-dried and thenstored at −20° C. For use, the plates are brought to room temperatureand blocked for nonspecific binding by addition of 150 microliters of 1%BSA and 1% PBS for 1 hour. The blocking material is discarded, and then100 microliters of solution of test serum diluted in 1% BSA/PBS is addedand incubated for 1 hour in a humidified box. After four washes with PBSand Tween, 100 microliter per well of a 1:1500 dilution of alkalinephosphatase-coupled goat antibuman gamma-chain specific antibody (Tago,Inc., Burlingame, Calif.) in 1% BSA/PBS is added per well for 1 hour.This antibody is discarded, and the wells are washed three times withPBS/Tween and four times with Tris/NaCl (0.05 mol/L of Tris base in 0.9N NaCl, pH 7.5). Substrate solution (disodium P-nitrophenol phosphate,1.5 mg/ml and 100 p.1 per well) is added, and color development isallowed to proceed until absorbance at 405 nm in the positive controlwells was 0.8. Levels are determined relative to a standard comprisingof pooled sera obtained from well-characterized pANCA ulcerative colitispatients. Results are expressed as ELISA units, which reflect apercentage of the standard. Sera with circulating antineutrophilcytoplasmic IgG antibody that is at or above the about 50 EU are termedANCA+. Optionally, the subject is treated with a non-anti-TNFtherapeutic agent provided herein. In some embodiments, the therapeuticagent comprises an anti-TL1A antibody.

Indirect immunofluorescence assay: Neutrophils separated by dextransedimentation are re-suspended in PBS, and 100,000 cells are prepared onslides by cytocentrifugation (Cytospin, Shandon Southern Products,Cheshire, U.K.). The slides are fixed in 100% methanol at 4° C. for 10minutes, air-dried, and stored at −20° C. The sera are tested at adilution of 1:20, and staining with fluoroscein-labeled F(ab′)₂gamma-chain-specific antibody is performed as described. Slides wereexamined by fluorescence microscopy (40×Epifluor lens, Carl Zeiss, Inc.,Thornwood, N.Y.).

Example 9

Phase 1 Clinical Trial

A phase 1 clinical trial is performed to evaluate the safety,tolerability, pharmacokinetics and pharmacodynamics of an anti-TL1Aantibody on subjects having an inflammatory disease or condition, orfibrostenotic and/or fibrotic disease.

Single ascending dose (SAD) arms: Subjects in each group (subjects aregrouped based on the presence of a level of ANCA at or above 100 ELISAunits (EU), or the presence of a level of ANCA below the 100 EU andabove 50 EU, and either of (i) a presence of the TNFRSF1B SNP, and (ii)decreased TNFR2 levels as compared in an individual who does not expressthe TNFRSF1B SNP) receive either a single dose of the antibody or aplacebo. Exemplary doses are 1, 3, 10, 30, 100, 300, 600 and 800 mg ofantibody. Safety monitoring and PK assessments are performed for apredetermined time. Based on evaluation of the PK data, and if theantibody is deemed to be well tolerated, dose escalation occurs, eitherwithin the same groups or a further group of healthy subjects. Doseescalation continues until the maximum dose has been attained unlesspredefined maximum exposure is reached or intolerable side effectsbecome apparent.

Multiple Ascending Dose (MAD) Arms:

Subjects in each group (subjects are grouped based on the same criteriaas above) receive multiple doses of the antibody or a placebo. The doselevels and dosing intervals are selected as those that are predicted tobe safe from the SAD data. Dose levels and dosing frequency are chosento achieve therapeutic drug levels within the systemic circulation thatare maintained at steady state for several days to allow appropriatesafety parameters to be monitored. Samples are collected and analyzed todetermination PK profiles.

Inclusion Criteria:

Healthy subjects of non-childbearing potential between the ages of 18and 55 years. Healthy is defined as no clinically relevant abnormalitiesidentified by a detailed medical history, full physical examination,including blood pressure and pulse rate measurement, 12 lead ECG andclinical laboratory tests. Female subjects of non-childbearing potentialmust meet at least one of the following criteria: (1) achievedpostmenopausal status, defined as: cessation of regular menses for atleast 12 consecutive months with no alternative pathological orphysiological cause; and have a serum follicle stimulating hormone (FSH)level within the laboratory's reference range for postmenopausalfemales; (2) have undergone a documented hysterectomy and/or bilateraloophorectomy; (3) have medically confirmed ovarian failure. All otherfemale subjects (including females with tubal ligations and females thatdo NOT have a documented hysterectomy, bilateral oophorectomy and/orovarian failure) will be considered to be of childbearing potential.Body Mass Index (BMI) of 17.5 to 30.5 kg/m2; and a total body weight >50kg (110 lbs). Evidence of a personally signed and dated informed consentdocument indicating that the subject (or a legal representative) hasbeen informed of all pertinent aspects of the study.

Inclusion Criteria:

Three groups of subjects are selected: (1) subjects having a presence ofthe level of ANCA at or above 100 EU, (2) subjects having a presence ofthe level of ANCA below 100 EU and above 50 EU and either (i) a presenceof the TNFRSF1B SNP, or (ii) decreased TNFR2 levels as compared in anindividual who does not express the TNFRSF1B SNP, and (3) subjectslacking a level of ANCA that is high related to a healthy individual.

Exclusion Criteria:

Evidence or history of clinically significant hematological, renal,endocrine, pulmonary, gastrointestinal, cardiovascular, hepatic,psychiatric, neurologic, or allergic disease (including drug allergies,but excluding untreated, asymptomatic, seasonal allergies at time ofdosing). Subjects with a history of or current positive results for anyof the following serological tests: Hepatitis B surface antigen (HBsAg),Hepatitis B core antibody (HBcAb), anti-Hepatitis C antibody (HCV Ab) orhuman immunodeficiency virus (HIV). Subjects with a history of allergicor anaphylactic reaction to a therapeutic drug. Treatment with aninvestigational drug within 30 days (or as determined by the localrequirement, whichever is longer) or 5 half-lives or 180 days forbiologics preceding the first dose of study medication. Pregnantfemales; breastfeeding females; and females of childbearing potential.

Primary Outcome Measures:

Incidence of dose limiting or intolerability treatment related adverseevents (AEs) [Time Frame: 12 weeks]. Incidence, severity and causalrelationship of treatment emergent AEs (TEAEs) and withdrawals due totreatment emergent adverse events [Time Frame: 12 weeks]. Incidence andmagnitude of abnormal laboratory findings [Time Frame: 12 weeks].Abnormal and clinically relevant changes in vital signs, blood pressure(BP) and electrocardiogram (ECG) parameters [Time Frame: 12 weeks].

Secondary Outcome Measures:

Single Ascending Dose: Maximum Observed Plasma Concentration (Cmax)[Time Frame: 12 weeks]. Single Ascending Dose: Time to Reach MaximumObserved Plasma Concentration (Tmax) [Time Frame: 12 weeks]. SingleAscending Dose: Area under the plasma concentration-time profile fromtime zero to 14 days (AUC14 days) [Time Frame: 12 weeks]. SingleAscending Dose: Area under the plasma concentration-time profile fromtime zero extrapolated to infinite time (AUCinf) [Time Frame: 12 weeks].Single Ascending Dose: Area under the plasma concentration-time profilefrom time zero to the time of last quantifiable concentration (AUClast)[Time Frame: 12 weeks]. Single Ascending Dose: Dose normalized maximumplasma concentration (Cmax[dn]) [Time Frame: 12 weeks]. Single AscendingDose: Dose normalized area under the plasma concentration-time profilefrom time zero extrapolated to infinite time (AUCinf[dn]) [Time Frame:12 weeks]. Single Ascending Dose: Dose normalized area under the plasmaconcentration-time profile from time zero to the time of lastquantifiable concentration (AUClast[dn]) [Time Frame: 12 weeks]. SingleAscending Dose: Plasma Decay Half-Life (t½) [Time Frame: 12 weeks].Plasma decay half-life is the time measured for the plasma concentrationto decrease by one half. Single Ascending Dose: Mean residence time(MRT) [Time Frame: 12 weeks]. Single Ascending Dose: Volume ofDistribution at Steady State (Vss) [Time Frame: 6 weeks]. Volume ofdistribution is defined as the theoretical volume in which the totalamount of drug would need to be uniformly distributed to produce thedesired blood concentration of a drug. Steady state volume ofdistribution (Vss) is the apparent volume of distribution atsteady-state. Single Ascending Dose: Systemic Clearance (CL) [TimeFrame: 6]. CL is a quantitative measure of the rate at which a drugsubstance is removed from the body.

Multiple Ascending Dose First Dose: Maximum Observed PlasmaConcentration (Cmax) [Time Frame: 12 weeks]. Multiple Ascending DoseFirst Dose: Time to Reach Maximum Observed Plasma Concentration (Tmax)[Time Frame: 12 weeks]. Multiple Ascending Dose First Dose: Area underthe plasma concentration-time profile from time zero to time τ, thedosing interval where τ=2 weeks (AUCτ) [Time Frame: 12 weeks]. MultipleAscending Dose First Dose: Dose normalized maximum plasma concentration(Cmax[dn]) [Time Frame: 12 weeks]. Multiple Ascending Dose First Dose:Dose normalized Area under the plasma concentration-time profile fromtime zero to time τ, the dosing interval where τ=2 weeks (AUCτ[dn])[Time Frame: 12 weeks]. Plasma Decay Half-Life (t½) [Time Frame: 12weeks]. Plasma decay half-life is the time measured for the plasmaconcentration to decrease by one half. Multiple Ascending Dose FirstDose: Mean residence time (MRT) [Time Frame: 12 weeks]. Apparent Volumeof Distribution (Vz/F) [Time Frame: 12 weeks]. Volume of distribution isdefined as the theoretical volume in which the total amount of drugwould need to be uniformly distributed to produce the desired plasmaconcentration of a drug. Apparent volume of distribution after oral dose(Vz/F) is influenced by the fraction absorbed. Multiple Ascending DoseFirst Dose: Volume of Distribution at Steady State (Vss) [Time Frame: 12weeks]. Volume of distribution is defined as the theoretical volume inwhich the total amount of drug would need to be uniformly distributed toproduce the desired blood concentration of a drug. Steady state volumeof distribution (Vss) is the apparent volume of distribution atsteady-state. Multiple Ascending Dose First Dose: Apparent OralClearance (CL/F) [Time Frame: 12 weeks]. Clearance of a drug is ameasure of the rate at which a drug is metabolized or eliminated bynormal biological processes. Clearance obtained after oral dose(apparent oral clearance) is influenced by the fraction of the doseabsorbed. Clearance is estimated from population pharmacokinetic (PK)modeling. Drug clearance is a quantitative measure of the rate at whicha drug substance is removed from the blood. Multiple Ascending DoseFirst Dose: Systemic Clearance (CL) [Time Frame: 12 weeks]. CL is aquantitative measure of the rate at which a drug substance is removedfrom the body.

Multiple Ascending Dose Multiple Dose: Maximum Observed PlasmaConcentration (Cmax) [Time Frame: 12 weeks]. Multiple Ascending DoseMultiple Dose: Time to Reach Maximum Observed Plasma Concentration(Tmax) [Time Frame: 12 weeks]. Multiple Ascending Dose Multiple Dose:Area under the plasma concentration-time profile from time zero to timeτ, the dosing interval where i=2 weeks (AUCτ) [Time Frame: 12 weeks].Multiple Ascending Dose Multiple Dose: Dose normalized maximum plasmaconcentration (Cmax[dn]) [Time Frame: 12 weeks]. Multiple Ascending DoseMultiple Dose: Dose normalized Area under the plasma concentration-timeprofile from time zero to time τ, the dosing interval where τ=2 weeks(AUCτ [dn]) [Time Frame: 12 weeks]. Multiple Ascending Dose MultipleDose: Plasma Decay Half-Life (t½) [Time Frame: 12 weeks]. Plasma decayhalf-life is the time measured for the plasma concentration to decreaseby one half. Multiple Ascending Dose Multiple Dose: Apparent Volume ofDistribution (Vz/F) [Time Frame: 12 weeks]. Volume of distribution isdefined as the theoretical volume in which the total amount of drugwould need to be uniformly distributed to produce the desired plasmaconcentration of a drug. Apparent volume of distribution after oral dose(Vz/F) is influenced by the fraction absorbed. Multiple Ascending DoseMultiple Dose: Volume of Distribution at Steady State (Vss) [Time Frame:12 weeks]. Volume of distribution is defined as the theoretical volumein which the total amount of drug would need to be uniformly distributedto produce the desired blood concentration of a drug. Steady statevolume of distribution (Vss) is the apparent volume of distribution atsteady-state.

Multiple Ascending Dose Multiple Dose: Apparent Oral Clearance (CL/F)[Time Frame: 12 weeks]. Clearance of a drug is a measure of the rate atwhich a drug is metabolized or eliminated by normal biologicalprocesses. Clearance obtained after oral dose (apparent oral clearance)is influenced by the fraction of the dose absorbed. Clearance wasestimated from population pharmacokinetic (PK) modeling. Drug clearanceis a quantitative measure of the rate at which a drug substance isremoved from the blood. Multiple Ascending Dose Multiple Dose: SystemicClearance (CL) [Time Frame: 12 weeks]. CL is a quantitative measure ofthe rate at which a drug substance is removed from the body. MultipleAscending Dose Multiple Dose: Minimum Observed Plasma TroughConcentration (Cmin) [Time Frame: 12 weeks]. Multiple Ascending DoseMultiple Dose: Average concentration at steady state (Cav) [Time Frame:12 weeks]. Multiple Ascending Dose Multiple Dose: Observed accumulationratio (Rac) [Time Frame: 12 weeks]. Multiple Ascending Dose MultipleDose: Peak to trough fluctuation (PTF) [Time Frame: 12 weeks]. MultipleAscending Dose Additional Parameter: estimate of bioavailability (F) forsubcutaneous administration at the corresponding intravenous dose [TimeFrame: 12 weeks]. Immunogenicity for both Single Ascending Dose andMultiple Ascending Dose: Development of anti-drug antibodies (ADA) [TimeFrame: 12 weeks].

Example 10

Phase 1B Clinical Trial

A phase 1b open label clinical trial is performed to evaluate efficacyof an anti-TL1A antibody on subjects having an inflammatory disease orcondition or fibrostenotic and/or fibrotic disease.

Arms:

5 patients positive a presence of the level of ANCA at or above 100ELISA units (EU) are administered the antibody. 5 patients positive fora presence of a level of ANCA below the 100 EU and above 50 EU andeither of (i) a presence of the TNFRSF1B SNP, and (ii) decreased TNFR2levels as compared in an individual who does not express the TNFRSF1BSNP, are administered the antibody. 5-10 patients negative for a highlevel of ANCA relative to a healthy individual are administered theantibody. Patients are monitored in real-time. Central ready ofendoscopy and biopsy is employed, with readers blinded to point of timeof treatment and endpoints.

Inclusion Criteria:

Three groups of subjects are selected: (1) subjects having a presence ofthe level of ANCA at or above 100 EU, (2) subjects having a presence ofthe level of ANCA below 100 EU and above 50 EU and either (i) a presenceof the TNFRSF1B SNP, or (ii) decreased TNFR2 levels as compared in anindividual who does not express the TNFRSF1B SNP, and (3) subjectslacking a level of ANCA that is high related to a healthy individual.

Primary Outcome Measures:

Simple Endoscopic Score for Crohn's Disease (SESCD), Crohn's DiseaseActivity Index (CDAI), and Patient Reported Outcome (PRO). If riskeither positive group shows 50% reduction from baseline, a Phase 2aclinical trial is performed.

Inclusion Criteria:

PRO entry criteria: Abdominal pain score of 2 or more and/or stoolfrequency score of 4 or more. Primary outcome would be pain core of 0 or1 and stool frequency score of 3 or less with no worsening frombaseline. Endoscopy entry criteria: SESCD ileum only entry at score of 4and 6 if colon is involved. Primary endoscopic outcome is 40-50% deltaof mean SESCD.

Example 11

Phase 2A Clinical Trial

A phase 2a clinical trial is performed to evaluate the efficacy of ananti-TL1A antibody in subjects having an inflammatory disease orcondition, or fibrostenotic and/or fibrotic disease.

Arms:

40 patients per arm (antibody and placebo arms) are treated withantibody or placebo for 12 weeks. An interim analysis is performed after20 patients from each group are treated at the highest dose to look fora 40-50% delta between placebo and treated group in primary outcome (50%reduction from baseline in SESCD, CDAI, and PRO).

Primary Outcome Measures:

Simple Endoscopic Score for Crohn's Disease (SESCD), Crohn's DiseaseActivity Index (CDAI), and Patient Reported Outcome (PRO).

Inclusion Criteria:

PRO entry criteria: Abdominal pain score of 2 or more and/or stoolfrequency score of 4 or more. Primary outcome would be pain core of 0 or1 and stool frequency score of 3 or less with no worsening frombaseline. Endoscopy entry criteria: SESCD ileum only entry at score of 4and 6 if colon is involved. Primary endoscopic outcome is 40-50% deltaof mean SESCD.

The various methods and techniques described above provide a number ofways to carry out the application. Of course, it is to be understoodthat not necessarily all objectives or advantages described can beachieved in accordance with any particular embodiment described herein.Thus, for example, those skilled in the art will recognize that themethods can be performed in a manner that achieves or optimizes oneadvantage or group of advantages as taught herein without necessarilyachieving other objectives or advantages as taught or suggested herein.A variety of alternatives are mentioned herein. It is to be understoodthat some preferred embodiments specifically include one, another, orseveral features, while others specifically exclude one, another, orseveral features, while still others mitigate a particular feature byinclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be employed invarious combinations by one of ordinary skill in this art to performmethods in accordance with the principles described herein. Among thevarious elements, features, and steps some will be specifically includedand others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the application extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses and modifications and equivalents thereof.

Preferred embodiments of this application are described herein,including the best mode known to the inventors for carrying out theapplication. Variations on those preferred embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. It is contemplated that skilled artisans canemploy such variations as appropriate, and the application can bepracticed otherwise than specifically described herein. Accordingly,many embodiments of this application include all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the application unless otherwise indicated herein orotherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that may have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

It is to be understood that the embodiments of the application disclosedherein are illustrative of the principles of the embodiments of theapplication. Other modifications that can be employed can be within thescope of the application. Thus, by way of example, but not oflimitation, alternative configurations of the embodiments of theapplication can be utilized in accordance with the teachings herein.Accordingly, embodiments of the present application are not limited tothat precisely as shown and described.

Various embodiments of the aspects disclosed herein are described abovein the Detailed Description. While these descriptions directly describethe above embodiments, it is understood that those skilled in the artmay conceive modifications and/or variations to the specific embodimentsshown and described herein. Any such modifications or variations thatfall within the purview of this description are intended to be includedtherein as well. Unless specifically noted, it is the intention of theinventors that the words and phrases in the specification and claims begiven the ordinary and accustomed meanings to those of ordinary skill inthe applicable art(s).

The foregoing description of various embodiments of the aspectsdisclosed herein known to the applicant at this time of filing theapplication has been presented and is intended for the purposes ofillustration and description. The present description is not intended tobe exhaustive nor limit the aspects disclosed herein to the precise formdisclosed and many modifications and variations are possible in thelight of the above teachings. The embodiments described serve to explainthe principles of the aspects disclosed herein and its practicalapplication and to enable others skilled in the art to utilize theaspects disclosed herein in various embodiments and with variousmodifications as are suited to the particular use contemplated.Therefore, it is intended that the aspects disclosed herein not belimited to the particular embodiments disclosed for carrying out theaspects disclosed herein.

While particular embodiments of the aspects disclosed herein have beenshown and described, it will be obvious to those skilled in the artthat, based upon the teachings herein, changes and modifications may bemade without departing from the aspects disclosed herein and its broaderaspects and, therefore, the appended claims are to encompass withintheir scope all such changes and modifications as are within the truespirit and scope of the aspects disclosed herein.

The invention claimed is:
 1. A method of treating a subject with an inflammatory, fibrostenotic, or fibrotic disease or condition, comprising administering to the subject a therapeutically effective amount of a therapeutic agent, provided a presence of a genetic risk variant at rs5745994 comprising a “C” allele at nucleobase position 76 within SEQ ID NO: 1 at a gene locus TNFRSF1B is detected in a biological sample obtained from the subject.
 2. The method of claim 1, wherein a decreased level of Tumor necrosis factor receptor 2 (TNFR2) is detected in a sample obtained from the subject, compared to a reference value obtained from an individual who is not a carrier of the genetic risk variant.
 3. The method of claim 1, wherein a level of ANCA is detected in a biological sample obtained from the subject that is at or above a first threshold level comprising about 100 ELISA units (EU).
 4. The method of claim 1, wherein a level of ANCA is detected in a biological sample obtained from the subject that is lower than a first threshold level comprising about 100 ELISA units (EU) and higher than a second threshold level comprising about 50 EU.
 5. The method of claim 4, wherein the level of ANCA and the presence of the risk variant is indicative that the subject is non-responsive, or susceptible to non-response, to anti-Tumor Necrosis Factor alpha (TNFα) therapy.
 6. The method of claim 4, wherein the level of ANCA, the presence of the genetic risk variant, and a decreased level of TNFR2 as compared to a reference value obtained from an individual who is not a carrier of the genetic risk variant, detected in the biological sample obtained from the subject is indicative that the subject is nonresponsive, or susceptible to non-response, to anti-TNFα therapy.
 7. The method of claim 1, wherein the presence of the genetic risk variant detected in the biological sample obtained from the subject is indicative that the subject is non-responsive, or susceptible to non-response, to anti-TNFα therapy.
 8. The method of claim 1, wherein a level of ANCA is detected in a biological sample obtained from the subject using an assay comprising an anti-ANCA antibody.
 9. The method of claim 1, wherein the inflammatory, fibrostenotic, or fibrotic disease or condition comprises inflammatory bowel disease (IBD), Crohn's disease (CD), perianal Crohn's disease (pCD), ulcerative colitis (UC), rheumatoid arthritis, multiple sclerosis, psoriasis, chronic colitis, pancreatitis, leukopenia, colonic fibrosis, primary sclerosing cholangitis, progressive systemic sclerosis, chronic asthma, or fibrostenosis of a small and/or large intestine, or a combination thereof.
 10. The method of claim 1, wherein the genetic risk variant is detected by contacting the biological sample obtained from the subject with a nucleic acid molecule comprising a nucleic acid sequence capable of hybridizing to 10 contiguous nucleobases of SEQ ID NO. 1 spanning nucleobase 76 within SEQ ID NO: 1 under standard hybridization conditions.
 11. The method of claim 10, wherein the standard hybridization conditions comprise an annealing temperature between about 30° C. and about 65° C.
 12. A method comprising: (a) assaying to detect in a biological sample obtained from a subject with an inflammatory, fibrostenotic, or fibrotic disease or condition, a level of antineutrophil cytoplasmic antibody (ANCA), a presence of a genetic risk variant comprising a risk allele at rs5745994 comprising a “C” allele at nucleobase position 76 within SEQ ID NO: 1 at a gene locus TNFRSF1B, or a combination thereof; (b) identifying the subject as being non-responsive to anti-TNFα therapy, or susceptible to non-response to anti-TNFα therapy, provided (i) a level of ANCA that is at or above a first threshold level comprising about 100 ELISA units (EU), or (ii) a level of ANCA that is lower than the first threshold level and above a second threshold level comprising about 50 EU, and the presence of the genetic risk variant, are detected in the biological sample obtained from the subject; and (c) treating, or prescribing a treatment for, the inflammatory, fibrostenotic, or fibrotic disease or condition in the subject, provided the subject is identified as being non-responsive to anti-TNFα therapy, or susceptible to non-response to anti-TNFα therapy.
 13. The method of claim 12, wherein treating the inflammatory, fibrostenotic, or fibrotic disease or condition in the subject of (c) further comprises administering to the subject a therapeutically effective amount of a therapeutic agent.
 14. The method of claim 12, wherein the inflammatory, fibrostenotic, or fibrotic disease or condition comprises inflammatory bowel disease (IBD), Crohn's disease (CD), perianal Crohn's disease (pCD), ulcerative colitis (UC), rheumatoid arthritis, multiple sclerosis, psoriasis, chronic colitis, pancreatitis, leukopenia, colonic fibrosis, primary sclerosing cholangitis, progressive systemic sclerosis, chronic asthma, or fibrostenosis of a small and/or large intestine, or a combination thereof.
 15. The method of claim 12, further comprising assaying to detect a level of TNFR2, wherein a decrease in TNFR2, compared to a reference value obtained from an individual who is not a carrier of the genetic risk variant, is indicative of non-response to anti-TNFα therapy in the subject.
 16. The method of claim 12, wherein the level of ANCA is detected using an assay comprising an anti-ANCA antibody.
 17. The method of claim 16, where the assay comprises a neutrophil-fixed enzyme-linked immunosorbent assay (neutrophil fixed-ELISA).
 18. The method of claim 12, wherein the genetic risk variant is detected by contacting the sample obtained from the subject with a nucleic acid molecule comprising a nucleic acid sequence capable of hybridizing to 10 contiguous nucleobases of SEQ ID NO. 1 spanning nucleobase 76 within SEQ ID NO: 1 under standard hybridization conditions, wherein the standard hybridization conditions comprise an annealing temperature between about 30° C. and about 65° C. 